N-(hydrophobe-substituted) vancosaminyl [Ψ-[C(=NH) NH] Tpg4] vancomycin and [Ψ-[CH2NH]Tpg4] vancomycin

ABSTRACT

The total synthesis and evaluation of key analogs of vancomycin containing single atom changes in the binding pocket are disclosed as well as their peripherally modified, N-(hydrophobe-substituted) derivatives exemplified by a N-4-(4′-chlorobiphenyl)-methyl derivative and their pharmaceutically acceptable salts are disclosed. Their evaluation indicates the combined pocket and peripherally modified analogs exhibit a remarkable spectrum of antimicrobial activity and truly impressive potencies against both vancomycin-sensitive and -resistant bacteria, and likely benefit from two independent and synergistic mechanisms of action. A pharmaceutical composition containing a contemplated compound or its pharmaceutically acceptable salt is disclosed, as is a method of treating a bacterial infection in a mammal by administering an antibacterial amount of a contemplated compound or its salt as above to an infected mammal in need of treatment.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority from provisional applications No.62/022,990, filed Jul. 10, 2014, and No. 62/109,405, filed Jan. 29,2015, whose disclosures are incorporated by reference.

GOVERNMENTAL SUPPORT

The present invention was made with governmental support pursuant togrant CA041101 from the National Institutes of Health/National CancerInstitute. The government has certain rights in the invention.

BACKGROUND ART

The glycopeptide antibiotics are among the most important class of drugsused in the treatment of resistant bacterial infections. [(a) Cooper etal., In Vancomycin, A Comprehensive Review of 30 Years of ClinicalExperience, 1986; pp 1-5, Park Row Publications, Indianapolis, Ind.; (b)Glycopeptide Antibiotics; Nagarajan, R., Ed.; Marcel Dekker: New York,1994; (c) Kahne et al., Chem. Rev. 2005, 105, 425.] Vancomycin[McCormick et al., Antibiot. Annu. 1955-1956, 606], teicoplanin [Parentiet al., J.

Antibiot. 1978, 31, 276] and a set of recently approved semisyntheticderivatives, including oritavancin (August 2014) [(a) Nicas et al.,Antimicrob. Agents Chemother. 1996, 40, 2194; (b) Nagarajan et al., J.Antibiot. 1989, 42, 63; (c) Markham, Drugs 2014, 74, 1823], dalbavancin(May 2014) [(a) Candiani net al., J. Antimicrob. Chemother. 1999, 44,179; (b) Anderson et al., Drugs 2008, 68, 639] and telavancin (September2009) [(a) Judice et al., Bioorg. Med. Chem. Lett. 2003, 13, 4165; (b)Corey et al., Nat. Rev. Drug Discovery 2009, 8, 929] are widely orincreasingly used to treat clinically refractory and resistant bacterialinfections.

Vancomycin, Compound 1, is the central member of the glycopeptideantibiotics that are among

the most important class of drugs used in the treatment of resistantbacterial infections. [(a) Glycopeptide Antibiotics; Nagarajan, R., Ed.;Marcel Dekker: New York, 1994; (b) Kahne et al., Chem. Rev. 2005, 105,425.] Although it was disclosed in 1956 [McCormick et al., Antibiot.Annu. 1955-1956, 606], and introduced into the clinic in 1958, thestructure of vancomycin was established only 25-30 years later (above).[Harris et al., J. Am. Chem. Soc. 1983, 105, 6915.]

After more than 50 years of clinical use and even with the additionalwidespread use of glycopeptide antibiotics for agricultural livestock(avoparcin), worldwide observation of vancomycin-resistant pathogens hasonly slowly emerged. This was first restricted to vancomycin-resistantEnterococci (VRE) initially detected in 1987 after 30 years of clinicaluse [(a) Leclercq et al., N. Engl. J. Med. 1988, 319, 157; (b)Courvalin, Clin. Infect. Dis. 2006, 42, S25] but recently includes themore feared emergence of vancomycin-resistant Staphylococcus aureus(VRSA) first detected in 2002. [(a) Weigel et al., Science 2003, 302,1569; (b) Howden et al., Clin. Microbiol. Rev. 2010, 23, 99; (c) Walshet al., Ann. Rev. Microbiol. 2002, 56, 657]. In spite of the increasingprevalence of VRE, such infections presently remain sensitive to othercommon antibiotic classes although a time may come when this will nolonger be the case.

More significant is the emergence of VRSA, which has already acquiredresistance to other common classes of antibiotics. Treatment options insuch cases are expected to be limited and, outside the new generationglycopeptide antibiotics, these presently include antibiotics known toeasily evoke resistance (linezolide, daptomycin). [(a) Brickner, Curr.Pharm. Des. 1996, 2, 175; (b) Scheetz et al., Antimicrob. AgentsChemother. 2008, 52, 2256; (c) Brickner et al., J. Med. Chem. 2008, 51,1981; and (a) Baltz et al., Nat. Prod. Rep. 2005, 22, 717; (b) Baltz,Curr. Opin. Chem. Biol. 2009, 13, 144] have been designated orrecommended for use as “reserve antibiotics”; ones that should beemployed sparingly to preserve their effectiveness as drugs of lastresort against intractable infections. This has intensified interest inthe development of alternative treatments for resistant pathogens thatdisplay the remarkable clinical durability of vancomycin [(a) Cooper etal., In Vancomycin, A Comprehensive Review of 30 Years of ClinicalExperience, 1986; pp 1-5, Park Row Publications, Indianapolis, Ind.; (b)Glycopeptide Antibiotics; Nagarajan, R., Ed.; Marcel Dekker: New York,1994; (c) Kahne et al., Chem. Rev. 2005, 105, 425; (d) Malabarba et al.,Med. Res. Rev. 1997, 17, 69; (e) Najarajan et al., Drugs 2004, 64, 913;(f) Butler et al., J. Antibiot. 2014, 67, 631].

Clinical uses of vancomycin include the treatment of patients ondialysis, allergic to β-lactam antibiotics, or undergoing cancerchemotherapy. [(a) Cooper et al., In Vancomycin, A Comprehensive Reviewof 30 Years of Clinical Experience, 1986; pp 1-5, Park Row Publications,Indianapolis, Ind. (b) Glycopeptide Antibiotics; Nagarajan, R., Ed.;Marcel Dekker: New York, 1994. (c) Kahne et al., Chem. Rev. 2005, 105,425.] However, the most widely recognized use of vancomycin is thetreatment of methicillin-resistant Staphylococcus aureus (MRSA)infections. [(a) Cooper et al., In Vancomycin, A Comprehensive Review of30 Years of Clinical Experience, 1986; pp 1-5, Park Row Publications,Indianapolis, Ind. (b) Glycopeptide Antibiotics; Nagarajan, R., Ed.;Marcel Dekker: New York, 1994. (c) Kahne et al., Chem. Rev. 2005, 105,425.] The prevalence of MRSA in intensive care units (ICU, 60% of SAinfections in the US are MRSA) [(a) CDC (2003); National NosocomialInfections Surveillance (NNIS) System Report, Data Summary from January1992 Through June 2004, Issued October 2004. Am. J. Infect. Control2004, 32, 470; (b) Laxminarayan, Antibiotic Resistance: The UnfoldingCrisis. In Extending the Cure, Policy Responses to the Growing Treat ofAntibiotic Resistance, Laxminarayan et al., Eds.; Resources for theFuture, 2007, Chapter 1, pp 25-37; (c) Walsh et al., Sci. Am. 2009, 301(1), 44] and its movement from a hospital-acquired to acommunity-acquired infection in the last 10 years has increased thenumber and intensified the need to treat such resistant bacterialinfections.

In addition, vancomycin-resistant bacterial strains are also on the risewith US ICU clinical isolates of vancomycin-resistant Enterococcusfaecalis (VRE) approaching 30% [(a) CDC (2003); National NosocomialInfections Surveillance (NNIS) System Report, Data Summary from January1992 Through June 2004, Issued October 2004. Am. J. Infect. Control2004, 32, 470; (b) Laxminarayan, Antibiotic Resistance: The UnfoldingCrisis. In Extending the Cure, Policy Responses to the Growing Treat ofAntibiotic Resistance, Laxminarayan et al., Eds.; Resources for theFuture, 2007, Chapter 1, pp 25-37; (c) Walsh et al., Sci. Am. 2009, 301(1), 44], albeit in strains presently sensitive to other antibiotics.Most feared is the recent emergence of MRSA strains now resistant orinsensitive to vancomycin (VRSA and VISA). This poses a major healthproblem and has intensified efforts to develop antibiotics to not onlycombat this resistance, but that also display the durability ofvancomycin [(a) Harris et al., J. Am. Chem. Soc. 1983, 105, 6915; (b)Williamson et al., J. Am. Chem. Soc. 1981, 103, 6580].

Vancomycin is structurally based on a heptapeptide scaffold that hasundergone extensive oxidative cross-linking. Five of the seven residuesare aromatic, and each residue is assigned a number in the sequence,beginning with leucine at position 1, and a hydroxyphenylglycine (HPG)at residue position 4.

As is seen from the structural formula above, vancomycin contains twoamine groups, a carboxylic acid and three potentially acidic phenolichydroxyl groups. Vancomycin is reported to have the following pKavalues: 7.75, 8.89 (amines; basic), 2.18 (carboxyl), 9.59, 10.4 and 12(phenolic; acidic) [Vijan, Rev. Roum. Chim. 2009, 54(10), 807-813].Vancomycin hydrochloride is sold for both oral and parenteraladministration.

After more than 50 years of clinical use and with the even morewidespread utilization of glycopeptide antibiotics for agriculturallivestock (avoparcin), worldwide observation of vancomycin-resistantpathogens has slowly emerged. This was first restricted tovancomycin-resistant Enterococci (VRE) [(a) Leclercq et al., N. Engl. J.Med. 1988, 319, 157; (b) Courvalin, Clin. Infect. Dis. 2006, 42, S25],but more recently includes the detection of vancomycin-resistantStaphylococcus aureus (VRSA) [(a) Weigel et al., Science 2003, 302,1569; (b) Walsh et al., Ann. Rev. Microbiol. 2002, 56, 657]. Interesthas consequently intensified in the development of alternativetreatments for resistant pathogens that display the remarkabledurability of vancomycin, including new derivatives of the glycopeptideantibiotics [(a) Glycopeptide Antibiotics; Nagarajan, R., Ed.; MarcelDekker: New York, 1994; (b) Kahne et al., Chem. Rev. 2005, 105, 425; (c)Malabarba et al., Med. Res. Rev. 1997, 17, 69; (d) Najarajan et al.,Drugs 2004, 64, 913; (e) Süssmuth, ChemBioChem 2002, 3, 295; (f) et al.,Chem. Rev. 2005, 105, 449; (g) von Nussbaum et al., Angew. Chem., Int.Ed. 2006, 45, 5072].

The clinical durability can be attributed to several complementaryfeatures of vancomycin that result in inhibition of bacterial cell wallbiosynthesis and its integrity. [James et al., ACS Chem. Biol. 2012, 7,797] Foremost of the features responsible for this durability is itsprimary biological target (binding to D-Ala-D-Ala). This target is notonly unique to bacteria, but it is also a structural component of thebacterial cell wall and a substrate for an enzymatic reaction. It is nota protein or nucleic acid target and, as a consequence, it is notsubject to alteration by genetic mutation. Moreover, the ramificationsof additional candidate binding sites within the bacterial cell wall(not only D-Ala-D-Ala, but also D-Ala-Gly and Gly-Gly) have yet to bedefined.

Vancomycin's primary mechanism of action involves substratesequestration (D-Ala-D-Ala) for a critical late-stage enzyme(transpeptidase) catalyzed reaction needed for peptidoglycancross-linking and bacterial cell wall maturation. However, it is thoughtto also inhibit transglycosylase-catalyzed incorporation of lipidintermediate II into the repeating polysaccharide backbone of thebacterial cell wall. With this second mechanism of action forvancomycin, it is not yet established whether this involves directbinding of the appended disaccharide to the enzyme active site, orwhether additional cell wall binding sites (e.g., D-Ala-D-Ala,D-Ala-Gly, or Gly-Gly) contribute to its localization and indirectenzyme inhibition. Because there may be two or more mechanisms of actionthat contribute to the inhibition of bacterial cell wall maturation byvancomycin, full bacterial resistance may require statistically unlikelysimultaneous changes to each to overcome all contributing mechanisms.

Just as importantly, the site of action is at the bacterial cell wallsurface and not at an intracellular target. As a result, no bacterialcell wall penetration or import mechanism is needed and this permitsvancomycin to avoid the common resistance mechanisms mediated by effluxpumps, blocked transport, and deactivation by cytosolic metabolicenzymes. [(a) Wright, Chem. Commun. 2011, 47, 4055; (b) Walsh, C. T.Nature, 2000, 406, 775]

Regardless of the origin and it is likely there are additional featurescontributing to the durability of vancomycin that are not yetrecognized, it is most revealing that the primary mechanism ofresistance to the glycopeptide antibiotics (VanA and VanB) wastransferred to pathogenic bacteria from non-pathogenic producingorganisms that use this inducible mechanism to protect themselves duringvancomycin production. [Marshall et al., Antimicrob. Agents Chemother.1998, 42, 2215] Significantly, this highlights that pathogenic bacteriahave not yet independently evolved effective resistance mechanisms tothe glycopeptide antibiotics even after more than 50 years of widespreaduse [identified mechanisms of resistance: VanA and VanB (inducibleD-Ala-D-Ala to D-Ala-D-Lac, 1000-fold), VanC (D-Ala-D-Ser, 20-fold), andthickened cell wall (increased number of target sites, 10-fold). See:Courvalin, Clin. Infect. Dis. 2006, 42, S25], suggesting thatfundamental solutions to VanA and VanB resistance may provide durableantibiotics with clinical lifetimes lasting 50 more years.

Due to their structural complexity, essentially all analogues of theglycopeptide antibiotics consist of semisynthetic derivatives of thenatural products. [(a) Cooper et al., In Vancomycin, A ComprehensiveReview of 30 Years of Clinical Experience, 1986; pp 1-5, Park RowPublications, Indianapolis, Ind.; (b) Glycopeptide Antibiotics;Nagarajan, R., Ed.; Marcel Dekker: New York, 1994; (c) Kahne et al.,Chem. Rev. 2005, 105, 425; (d) Malabarba, et al., Med. Res. Rev. 1997,17, 69; (e) Najarajan, J. Antibiot. 1993, 46, 1181; (f) Van Bambeke etal., Drugs 2004, 64, 913; (g) Butler et al., J. Antibiot. 2014, 67, 631]The most significant of the modifications introduce peripheralhydrophobic groups and these are found in each of the clinicallyapproved semisynthetic derivatives oritavancin, dalbavancin andtelavancin, whose structural formulas are shown below.

For both dalbavancin and telavancin, the long chain hydrophobic alkylchains are thought to provide selective membrane anchoring propertiesand promote antibiotic dimerization without impacting binding affinityto the primary biological target D-Ala-D-Ala. [a) Allen et al.,Antimicrob. Agents Chemother. 1996, 40, 2356; (b) Sharman et al., J. Am.Chem. Soc. 1997, 119, 12041; (c) Allen et al., FEMS Microbiol. Rev.2003, 26, 511] It is possible such semisynthetic changes to theglycopeptide antibiotics also avoid bacterial sensing of the antibioticchallenge and this may account for their VanB VRE activity firstobserved with teicoplanin. [(a) Hong et al., Adv. Exp. Med. Biol. 2008,631, 200; (b) Koteva et al., Nat. Chem. Biol. 2010, 6, 327; (c) Ikeda etal., J. Antibiot. 2010, 63, 533; (d) Kwun et al., Antimicrob. AgentsChemother. 2013, 57, 4470] Additionally, telavancin has been shown tofunction not only through the traditional mechanism of inhibition ofcell wall synthesis by binding D-Ala-D-Ala, but also through thedisruption of bacterial membrane integrity, a mechanism typically notobserved for the glycopeptide antibiotics. [Higgins et al., Antimicrob.Agents Chemother. 2005, 49, 1127]

One of the most widely recognized modifications is the 4-chlorobiphenylsubstitution of a peripheral carbohydrate. This substitution has beenexamined at range of positions in a variety of glycopeptide antibiotics,most notably in oritavancin [(a) Nicas et al., Antimicrob. AgentsChemother. 1996, 40, 2194; (b) Nagarajan et al., J. Antibiot. 1989, 42,63. (b) Markham, A. Drugs 2014, 74, 1823], theN-(4-chlorobiphenyl)methyl derivative of chloroeremomycin, and withvancomycin itself (CBP-vancomycin). [Cooper et al., J. Antibiot. 1996,49, 575]

In addition to promoting antibiotic dimerization, membrane anchoring,disruption of bacterial membrane integrity, and potentially avoidingbacterial sensing of the antibiotic challenge, the unique placement ofthe 4-chlorobiphenyl substituent introduces or potentiates a secondmechanism of action. The direct inhibition of transglycosylases mediatedby the modified carbohydrate has been identified as a second, noweffective, mechanism by which oritavancin exhibits antimicrobialactivity. [(a) Ge et al., Science 1999, 284, 507; (b) Chen et al., Proc.Natl. Acad. Sci. USA 2003, 100, 5658; (c) Goldman et al., Microbiol.Lett. 2000, 183, 209]

Regardless of the origin of the effects, such derivatives often increaseantibiotic potency as much as 100-fold. Although increasing bacterialsensitivity to the antibiotics, VanA vancomycin-resistant bacterialstrains (MIC=about 10 μg/mL) remain 1000-fold less sensitive thansusceptible strains (MIC=about 0.01 μg/mL). This suggested thatcombining such peripheral hydrophobic substitutions with vancomycinbinding pocket modifications that maintain D-Ala-D-Ala binding andreinstate binding to D-Ala-D-Lac would further increase theirantimicrobial activity against not only sensitive, but alsovancomycin-resistant bacteria to truly remarkable potencies.

Recently, and in an extension of work first directed at the totalsyntheses of the naturally occurring glycopeptide antibiotics [(a) Bogeret al., J. Am. Chem. Soc. 1999, 121, 3226; (b) Boger et al., J. Am.Chem. Soc. 1999, 121, 10004; (c) Boger et al., J. Am. Chem. Soc. 2000,122, 7416; (d) Boger et al., J. Am. Chem. Soc. 2001, 123, 1862; (e)Crowley et al., J. Am. Chem. Soc. 2004, 126, 4310; (f) Garfunkle et al.,J. Am. Chem. Soc. 2009, 131, 16036; (g) Shimamura et al., J. Am. Chem.Soc. 2010, 132, 7776; (h) Breazzano et al., J. Am. Chem. Soc. 2011, 133,18495; (i) James et al., ACS Chem. Biol. 2012, 7, 797; (j) Boger, Med.Res. Rev. 2001, 21, 356; (k) Evans et al., Angew. Chem., Int. Ed. 1998,37, 2700; (l) Evans et al., Angew. Chem., Int. Ed. 1998, 37, 2704; (m)Evans et al., J. Am. Chem. Soc. 1997, 119, 3419; (n) Evans et al., J.Am. Chem. Soc. 1997, 119, 3417; (o) Nicolaou et al., Angew. Chem., Int.Ed. 1998, 37, 2717; (p) Nicolaou et al., M. Angew. Chem., Int. Ed. 1998,37, 2708; (q) Nicolaou et al., Angew. Chem., Int. Ed. 1998, 37, 2714;(r) Boger, Med. Res. Rev. 2001, 21, 356; (s) Nicolaou et al., Angew.Chem., Int. Ed. 1999, 38, 2096; (t) Evans et al., Drugs Pharm. Sci.1994, 63, 63] the present inventor and co-workers described studies onthe binding pocket redesign of vancomycin [James et al., ACS Chem. Biol.2012, 7, 797] that are the first to directly address the molecular basisof clinical resistance to vancomycin. [(a) Bugg et al., Biochemistry1991, 30, 10408; Reviews: (b) Walsh, Science 1993, 261, 308; (c) Walshet al., Chem. Biol. 1996, 3, 21; (d) Lessard et al., Proc. Natl. Acad.Sci. USA 1999, 96, 11028; (e) Healy et al., Chem. Biol. 2000, 7, R109;(f) Perkins, Pharmacol. Ther. 1982, 16, 181; (g) Williams et al., J. Am.Chem. Soc. 1983, 105, 1332; (h) Schaefer et al., Structure 1996, 4,1509]

The destabilized binding to D-Ala-D-Lac is due to a combination of theloss of a H-bond central to ligand binding the antibiotic (10-fold), andan even more significant destabilizing lone pair repulsion between thevancomycin residue 4 carbonyl and D-Ala-D-Lac ester oxygens (100-fold).[McComas et al., J. Am. Chem. Soc. 2003, 125, 9314] The elucidation ofthis inducible mechanism of resistance (VanA and VanB) acquired fromnon-pathogenic vancomycin-producing organisms [Marshall et al.,Antimicrob. Agents Chemother. 1998, 42, 2215] also highlighted that suchvancomycin binding pocket modifications must target compounds that notonly establish binding to D-Ala-D-Lac, but that also maintainD-Ala-D-Ala binding. That targeting not only insures antimicrobialactivity against vancomycin-resistant bacteria (VanA and VanB), butadditionally assures maintained activity against vancomycin-sensitivebacteria.

Previous studies of the inventor and co-workers provided[Ψ[CH₂NH]Tpg⁴]vancomycin aglycon (Compound 10) [Crowley et al., Am.Chem. Soc. 2006, 128, 2885], which displayed such dual bindingproperties by virtue of removal of the lone pair repulsion between thevancomycin residue 4 carbonyl and D-Ala-D-Lac ester oxygens. This changereinstated commensurate activity against VanA VRE, validated theopportunities of the approach, and entailed removal of a single atomfrom the vancomycin binding pocket.

These efforts were followed by the total synthesis of[Ψ[C(═NH)NH]Tpg⁴]vancomycin aglycon (Compound 9) [(a) Xie et al., J. Am.Chem. Soc. 2011, 133, 13946; and (b) Xie et al., J. Am. Chem. Soc. 2012,134, 1284], providing a modified antibiotic that not only maintainedvancomycin's ability to bind the unaltered peptidoglycan D-Ala-D-Ala,but that also bound the altered ligand D-Ala-D-Lac just as effectivelyby virtue of its ability to serve as either a H-bond donor (forD-Ala-D-Lac) or H-bond acceptor (for D-Ala-D-Ala). Whereas the formerentails binding of the protonated amidine with D-Ala-D-Lac and replacesthe destabilizing carbonyl lone pair interaction with the ester oxygenlone pair with a stabilizing electrostatic interaction and perhaps areversed H-bond, the latter entails binding of D-Ala-D-Ala with theunprotonated amidine serving as a H-bond acceptor. [Okano et al., J. Am,Chem. Soc. 2012, 134, 8790] Not only did amidine Compound 9 displaybalanced binding affinity for both target ligands within 2-fold of thatwhich vancomycin aglycon exhibits with D-Ala-D-Ala, but it alsoexhibited effective antimicrobial activity against VanA VRE, beingequipotent to the activity that vancomycin displays against sensitivebacterial strains.

These latter studies represented the replacement of a single atom in thebinding pocket of the antibiotic aglycon (O→NH) to counter acomplementary exchange in the cell wall precursors of resistant bacteria(NH→O). Just as remarkable, it was established that[Ψ[C(═S)NH]Tpg⁴]vancomycin aglycon (Compound 8), which served as thepenultimate precursor to Compound 9 [(a) Xie et al., J. Am. Chem. Soc.2011, 133, 13946; (b) Xie et al., J. Am. Chem. Soc. 2012, 134, 1284],fails to bind D-Ala-D-Ala or D-Ala-D-Lac to any appreciable extent andis inactive against both vancomycin-sensitive and vancomycin-resistantbacteria.

The expectedly benign conversion of the residue 4 amide to a thioamidewith the exchange of a single atom in the binding pocket (O→S) provedsufficient to completely disrupt ligand binding. This loss in affinitywas attributed largely to the increased thiocarbonyl bond length andsize of the sulfur atom that are sufficient to sterically displace theligand out of the binding pocket and completely disrupt the intricatebinding of D-Ala-D-Ala. Significantly, the comparison of Compound 8 withCompound 9 highlighted just how remarkable the behavior of the amidineCompound 9 is. These aglycon structures and data are shown below.

ligand, K_(a) (M⁻¹) VanA^(a) compound 11, Y = NH 12, Y = O K_(a)(11/12)MIC, μg/mL  7, X = O 1.7 × 10⁵ 1.2 × 10² 1400    640  8, X = S 1.7 × 10²1.1 × 10¹ — >640   9, X = NH 7.3 × 10⁴ 6.9 × 10⁴   1.05    1^(b) 10, X =H₂ 4.8 × 10³ 5.2 × 10³  0.9   31 ^(a)Minimum inhibitory conc., E.faecalis (BM4166, VanA VRE). ^(b)Tested herein alongside Compounds 1-6.

The glycopeptide antibiotics inhibit bacterial cell wall synthesis bybinding the precursor peptidoglycan peptide terminus D-Ala-D-Ala,inhibiting transpeptidase-catalyzed cell wall cross-linking andmaturation [(a) Perkins, Pharmacol. Ther. 1982, 16, 181; (b) Williams etal., J. Am. Chem. Soc. 1983, 105, 1332; (c) Schaefer et al., Structure1996, 4, 1509].

In the clinically prominent resistant phenotypes, VanA and VanB,synthesis of the precursor lipid intermediates I and II continuecomplete with their pendant N-terminal D-Ala-D-Ala, but resistantbacteria sense the antibiotic challenge [(a) Hong et al., J. Adv. Exp.Med. Biol. 2008, 631, 200; (b) Koteva et al., Nat. Chem. Biol. 2010, 6,327; (c) Ikeda et al., J. Antibiot. 2010, 63, 533; (d) Kwun et al.,Antimicrob. Agents Chemother. 2013, 57, 4470] and initiate a late stageremodeling of their peptidoglycan termini from D-Ala-D-Ala toD-Ala-D-Lac [(a) Bugg et al., Biochemistry 1991, 30, 10408; (b) Walsh,Science 1993, 261, 308] to avoid the antibiotic action.

Through use of a two-component cell surface receptor sensing andsubsequent intracellular signaling system [(a) Hong et al., Adv. Exp.Med. Biol. 2008, 631, 200; (b) Koteva et al., Nat. Chem. Biol. 2010, 6,327; (c) Ikeda et al., J. Antibiot. 2010, 63, 533; (d) Kwun et al.,Antimicrob. Agents Chemother. 2013, 57, 4470], resistant bacteriainitiate a late stage remodeling of their peptidoglycan termini fromD-Ala-D-Ala to D-Ala-D-Lac [(a) Bugg et al., Biochemistry 1991, 30,10408; Reviews: (b) Walsh, Science 1993, 261, 308; (c) Walsh et al.,Chem. Biol. 1996, 3, 21; (d) Lessard et al., Proc. Natl. Acad. Sci. USA1999, 96, 11028; (e) Healy et al., Chem. Biol. 2000, 7, R109; (f)Perkins, Pharmacol. Ther. 1982, 16, 181; (g) Williams et al., J. Am.Chem. Soc. 1983, 105, 1332; (h) Schaefer et al., Structure 1996, 4,1509] to avoid the action of the antibiotic. The vancomycin bindingaffinity for this altered ligand is reduced 1000-fold [(a) Bugg et al.,Biochemistry 1991, 30, 10408; Reviews: (b) Walsh, Science 1993, 261,308; (c) Walsh et al., Chem. Biol. 1996, 3, 21; (d) Lessard et al.,Proc. Natl. Acad. Sci. USA 1999, 96, 11028; (e) Healy et al., Chem.Biol. 2000, 7, R109; (f) Perkins, Pharmacol. Ther. 1982, 16, 181; (g)Williams et al., J. Am. Chem. Soc. 1983, 105, 1332; (h) Schaefer et al.,Structure 1996, 4, 1509] resulting in a corresponding 1000-fold loss inantimicrobial activity.

The direct inhibition of transglycosylases mediated by aglycopeptide-modified carbohydrate has been implicated as a secondmechanism by which the lipophilic glycopeptides with impairedD-Ala-D-Lac or D-Ala-D-Ala binding properties exhibit antimicrobialeffects [(a) Ge et al., Science 1999, 284, 507; (b) Chen et al., Proc.Natl. Acad. Sci. USA 2003, 100, 5658]. Other compounds, includingtelavancin, have been shown to function both through the traditionalmechanism of inhibition of cell wall synthesis by binding to D-Ala-D-Alaand also through the disruption of bacterial membrane integrity, amechanism typically not observed for glycopeptides [(a) Higgins et al.,Antimicrob. Agents Chemother. 2005, 49, 1127; (b) Corey et al., Nat.Rev. Drug Discovery 2009, 8, 929].

Regardless of the origin of the effect, such derivatives typicallyincrease antibiotic potency as much as 100-fold. While increasingbacterial sensitivity to the antibiotics, VanA vancomycin-resistantbacterial strains (MIC=about 10 μg/mL) remain 1000-fold less sensitivethan susceptible strains (MIC=about 0.01 μg/mL).

Because of their structural complexity, essentially all new analogs ofthe glycopeptide antibiotics consist of semisynthetic derivatives of thenatural products [(a) Glycopeptide Antibiotics; Nagarajan, R., Ed.;Marcel Dekker: New York, 1994; (b) Kahne et al., Chem. Rev. 2005, 105,425; (c) Malabarba et al., Med. Res. Rev. 1997, 17, 69; (d) Najarajan etal., Drugs 2004, 64, 913]. The most significant of the modificationsintroduce peripheral hydrophobic groups into the glycopeptide structure.

Among the early-reported successes were those of Nagarajan et al., J.Antibiot. 1989, 42, 63 and Nagarajan, R. J. Antibiot. 1993, 46, 118 whodisclosed N-decyl, N-p-octylbenzyl and N-p-octyl-oxybenzyl groups bondedto the 4-epi-vancosaminyl substituent of a glycopeptide antibioticprovided enhanced potency against both sensitive and resistantEnterococci. See also, U.S. Pat. Nos. 5,840,684 and 5,843,889.

One later-developed, hydrophobe-modified glycopeptide is telavancin[Corey et al., Nat. Rev. Drug Discovery 2009, 8, 929-930; formerlyreferred to as TD-6424] a clinically approved (2009) semisyntheticderivative of vancomycin used to treat complicated skin infections thatare suspected or confirmed to be MRSA. This drug bears a hydrophobicN-ethylene-2-amino-N-decyl group bonded to the vancosaminyl nitrogen togrant increased activity against resistant organisms and a hydrophilicphosphonic acid side chain that provides improved pharmacokineticproperties. Oritavancin is another widely studiedhydrophobically-substituted vancomycin-like glycopeptide derivative thatcontains a N-4-(4′-chlorobiphenyl)methyl group bonded to the aminonitrogen of a L-4-epi-vancosaminyl-1,2-D-glucoside [(a) Malabarba etal., Med. Res. Rev. 1997, 17, 69; (b) Najarajan et al., Drugs 2004, 64,913], and a second L-4-epi-vancosaminyl substituent bonded to the cycliccore.

Dalbavancin and teicoplanin are other hydrophobe-substitutedglycopeptide antibiotics. Teicoplanin is a natural product that containsa heptapeptide cyclic core structure similar to that of vancomycin butcontains four internal cross-links as compared to the three cross-linkspresent in vancomycin. Teicoplanin also contains anN-acetyl-β-D-glucosamine and a D-mannose group separately bonded to thecyclic structure, as well as one of at least five differentC₁₀₋₁₁-acyl-β-D-glucosamine groups. Dalbavancin contains a cyclic core(scaffold) structure slightly different from teicoplanin, as well as acarboxyl-substituted, N—C₁₀-amidohexoside group, a3-(dimethylaminopropyl)amido group and a D-mannose group that are bondedto the cyclic core, but lacks an N-acetyl-β-D-glucosamine group presentin teicoplanin.

These hydrophobic modifications have been explored in a variety ofglycopeptide antibiotics and at range of positions, most notably inoritavancin [(a) Nicas et al., Antimicrob. Agents Chemother. 1996, 40,2194; (b) Nagarajan et al., J. Antibiot. 1989, 42, 63], theN-(4-chlorobiphenyl)methyl derivative of chloroeremomycin, and withvancomycin itself (Compound 4, CBP-vancomycin) [Kahne et al., Chem. Rev.2005, 105, 425]. Oritavancin, dalbavancin, teicoplanin, telavancin andsimilar glycopeptide antibiotics on which these modifications have beentried all have one or more of different glycosyl groups, different sidechain substituents, or one or more additional glycosyl groups comparedto vancomycin.

Kahne et al., Chem. Rev. 2005, 105, 425 reported that the minimuminhibitory concentration (MIC) against vancomycin-sensitive and-resistant strains of E. faecium of vancomycin itself and CBP-vancomycinwere 1 and 2048 μg/mL (vancomycin) vs. 0.03 and 16 μg/mL(CBP-vancomycin). Thus, the activity increased in the presence of theCBP group, but the vancomycin-resistant strain was still about 500-timesless sensitive than the sensitive strain.

Studies on the mechanism of action and have shown that theN-4-(4′-chlorobiphenyl)methyl side chain promotes antibioticdimerization and membrane anchoring and establishes antimicrobialactivity against vancomycin-resistant organisms despite a lack ofimproved binding with either D-Ala-D-Ala or D-Ala-D-Lac [(a) Allen etal., Antimicrob. Agents Chemother. 1996, 40, 2356; (b) Sharman et al.,J. Am. Chem. Soc. 1997, 119, 12041; (c) Allen et al., FEMS Microbiol.Rev. 2003, 26, 511]. It is possible such semisynthetic changes tovancomycin also avoid bacterial sensing of the antibiotic challenge andthis may account for their VanB VRE activity (like teicoplanin) [(a)Hong et al., J. Adv. Exp. Med. Biol. 2008, 631, 200; (b) Koteva et al.,Nat. Chem. Biol. 2010, 6, 327; (c) Ikeda et al., J. Antibiot. 2010, 63,533; (d) Kwun et al., Antimicrob. Agents Chemother. 2013, 57, 4470], orthat they may entail a second mechanism of action.

The full details of the total synthesis of the recently disclosed [Okanoet al., J. Am. Chem. Soc. 2014, 136, 13522] [Ψ[C(═NH)NH]Tpg⁴]vancomycinand [Ψ[C(═S)NH]Tpg⁴]-vancomycin, and their (4-chloro-biphenyl)methylderivatives are provided hereinafter. Analogous and previouslyunreported studies first developed with their corresponding syntheticC-terminal hydroxymethyl precursors, as well as the total synthesis of[Ψ[CH₂NH]Tpg⁴]vancomycin and their corresponding(4-chlorobiphenyl)methyl derivatives are also reported. The latterpreviously undisclosed studies complete an initial series of totallysynthetic vancomycin analogs bearing the peripheralL-vancosaminyl-1,2-D-glucosyl disaccharide as well as their(4-chlorobiphenyl)methyl derivatives.

Collectively, the compounds represent a key set of analogues ofvancomycin and its (4-chloro-biphenyl)methyl derivative containingsingle atom changes in the binding pocket. Their assessments indicatethat combined pocket and chiorobiphenyl (CBP) peripherally modifiedanalogues exhibit a remarkable spectrum of antimicrobial activity (VSSA,MRSA, VanA and VanB VRE) and impressive potencies against bothvancomycin-sensitive and vancomycin-resistant bacteria, and likelybenefit from two independent and synergistic mechanisms of action. Likevancomycin, such analogues are likely to display especially durableantibiotic activity not prone to rapidly acquired clinical resistance.

BRIEF SUMMARY OF THE INVENTION

The present invention provides an extension of studies on the totalsynthesis and evaluation of [Ψ[C(═NH) NH]Tpg⁴] vancomycin and relatedcompounds with introduction of the L-vancosaminyl-1,2-D-glucosyldisaccharide, and a hydrophobic N-vancosaminyl-substituted grouprepresenting a binding pocket analog of vancomycin itself containing abinding pocket single atom change, as well as the total synthesis andevaluation of [Ψ[CH₂NH]Tpg⁴]-vancomycin with introduction of theL-vancosaminyl-1,2-D-glucosyl disaccharide, and a hydrophobicN-vancosaminyl-substituted group. Although the attached carbohydrate invancomycin does not impact in vitro antimicrobial activity or influencetarget D-Ala-D-Ala or D-Ala-D-Lac binding affinities, the appendedcarbohydrate impacts in vivo activity, increasing water solubility,influencing the pharmacokinetics (PK) and distribution properties, andcontributing what is understood to be a second mechanism ofantimicrobial action.

Given the distinct origins of their impact on the antimicrobial activityof vancomycin, it was believed that incorporation of peripheralhydrophobic modifications into the structure of a bindingpocket-modified vancomycin would further increase the antimicrobialactivity of such a compound against not only sensitive, but alsovancomycin-resistant bacteria to provide truly enhanced potencies. Asidefrom the merits of such molecules as new therapeutics, their increasedpotencies would have the additional impact of reducing the amounts ofmaterial needed for preclinical exploration.

Although such a comparison could conceivably be demonstrated byO-substitution of a hydrophobe such as a 4-(4′-chlorobiphenyl)methylgroup to the synthetic aglycon Compound 9 on which original work wasconducted, the most definitive assessment of the dual impact is a directcomparison of a N-hydrophobe-substituted vancomycin such asN-4-(4′-chlorobiphenyl)methyl vancomycin (Compound 4; 4-CBP vancomycin)with the corresponding amidine-substituted Compound 6, wherein a singleatom exchange in the binding pocket was introduced, despite thesynthetic challenges this posed.

The total syntheses of [Ψ[C(═NH)NH]Tpg⁴]-vancomycin aglycon (Compound 9)from synthetic [Ψ[C(═S)NH]Tpg⁴]vancomycin aglycon (Compound 8) werepreviously disclosed [(a) Xie et al., J. Am. Chem. Soc. 2011, 133,13946; (b) Xie et al., J. Am. Chem. Soc. 2012, 134, 1284]. The totalsyntheses of [Ψ[C(═S)NH]Tpg⁴]vancomycin (Compound 2) and[Ψ[C(═NH)NH]Tpg⁴]vancomycin (Compound 3), as well as the syntheses ofillustrative N-hydrophobe-substituted vancomycins,N-4-(4′-chlorobiphenyl)-methyl [Ψ[C(═S)NH]Tpg⁴]vancomycin, Compound 5,and N-4-(4′-chlorobiphenyl)methyl [Ψ[C(═NH)NH]Tpg⁴]-vancomycin, Compound6, are disclosed herein. The antibacterial use of these compoundsparticularly against Gram-positive bacteria including bothvancomycin-sensitive and -resistant Staphylococcus aureus strains, andmethicillin-resistant Staphylococcus aureus (MRSA) is also disclosed.

Thus, one aspect of the invention contemplates a compound thatcorresponds in structure to that shown in Formula I, below, or itspharmaceutically acceptable salt,

wherein

X═H₂, S or NH; and

R is selected from the group consisting of H, (C₁-C₁₆)hydrocarbyl,aryl(C₁-C₆)-hydrocarbyldiyl, heteroaryl-(C₁-C₆)hydrocarbyldiyl,(C₁-C₆)hydrocarbyldiylheteroaryl, halo(C₁-C₁₂)-hydrocarbyldiyl, and(C₁-C₁₆)amido substituents, wherein an aryl or heteroaryl group isitself optionally substituted with up to three substituentsindependently selected from the group consisting of:

(i) hydroxy,

(ii) halo,

(iii) nitro,

(iv) (C₁-C₆)hydrocarbyl,

(v) halo(C₁-C₁₆)hydrocarbyl,

(vi) (C₁-C₆)hydrocarbyloxy,

(vii) halo(C₁-C₆)hydrocarbyloxy,

(viii) aryl, and

(ix) aryloxy, wherein an aryl or aryloxy substituent can itself besubstituted with up to three substituents independently selected fromthe group consisting of:

-   -   (i) hydroxy,    -   (ii) halo,    -   (iii) nitro,    -   (iv) (C₁-C₆)hydrocarbyl,    -   (v) halo(C₁-C₁₆)hydrocarbyl,    -   (vi) (C₁-C₆)hydrocarbyloxy, and    -   (vii) halo(C₁-C₆)hydrocarbyloxy; and        R¹ is CH₂OH, CH₂OR², where R² is (C₁-C₇)hydrocarboyl, C(O)OH        [carboxyl], C(O)R³, where R³ is (C₁-C₆)hydrocarbyloxy, or R³ is        NR⁴R⁵ where R⁴ and R⁵ are independently the same or different        and are H (hydrido), (C₁-C₆)hydrocarbyl or R⁴ and R⁵ together        with the depicted nitrogen atom form a 5-7 membered ring that        can contain one ring oxygen atom.

In some preferred embodiments, R is other than hydrido. In preferredembodiments, R¹ is CH₂OH (hydroxymethyl) or carboxyl. A compound inwhich R¹ is carboxyl is particularly preferred, and such a compound isoften used herein as illustrative of the compounds in which R¹ is asdescribed above.

One particularly preferred compound of Formula I is a 4-thioamido{Tpg⁴-thioamido; [C(═S)NH]} compound that corresponds in structure toFormula II, below,

wherein R and R¹ are as described above.

A still more particularly preferred compound of Formula I is a 4-amidino{Tpg⁴-amidino; [C(═NH)NH]} compound that corresponds in structure toFormula III, below,

wherein R and R¹ are also as described above.

Another particularly preferred compound of Formula I corresponds instructure to Formula IV, below,

wherein R and R¹ are also as described above.

Compounds 5, 6 and 17, whose structural formulas are shown below, arethe currently most

preferred antimicrobial compounds contemplated herein. Compounds 2(thioamide vancomycin), 3 (amidino vancomycin) and 16 [[Ψ[CH₂NH]Tpg⁴]-vancomycin], whose structural formulas are shown below, areimportant intermediates in the formation

of Compounds 5, 6 and 17, respectively, and also exhibit antimicrobialactivity.

Compounds 26, 27 and 28, whose structural formulas are shown below, arealso preferred antimicrobial compounds that compounds exhibitantimicrobial activity.

Another aspect of the invention is a pharmaceutical composition thatcontains an antimicrobial amount of a compound of Formulas I, II, III orIV, or a pharmaceutically acceptable salt thereof dissolved or dispersedin a physiologically (pharmaceutically) acceptable diluent (or carrier)is also contemplated. A particularly preferred compound of such acomposition is Compound 6.

A further aspect of the invention is a method of treating a mammalinfected with a bacterial infection, typically a Gram-positiveinfection, and therefore in need of antibacterial (antimicrobial)treatment. In accordance with a contemplated method, anantibacterial-effective amount of a compound of Formulas I, II, III orIV, such as Compound 6, or a pharmaceutically acceptable salt thereof isadministered to such an infected mammal in need. The administration isrepeated until the infection is diminished to a desired extent.

Definitions

In the context of the present invention and the associated claims, thefollowing terms have the following meanings:

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The words “ortho”, “meta” and “para” [“o”, “m”, and “p”] are used intheir usual manner to describe benzenoid compounds that are substituted“1-2”, “1-3” and “1-4”, respectively. Those same words are also usedherein as a convenience to describe those same substitution patterns inaliphatic compounds.

The word “hydrocarbyl” is used herein as a short hand term for anon-aromatic group that includes straight and branched chain aliphaticas well as alicyclic groups or radicals that contain only carbon andhydrogen. Thus, alkyl, alkenyl and alkynyl groups are contemplated,whereas aromatic hydrocarbons such as phenyl and naphthyl groups, whichstrictly speaking are also hydrocarbyl groups, are referred to herein asaryl groups or radicals, as discussed hereinafter.

Where a specific aliphatic hydrocarbyl substituent group is intended,that group is recited; i.e., C₁-C₄ alkyl, methyl or tert-butyl.Exemplary hydrocarbyl groups contain a chain of 1 to 4 carbon atoms, andpreferably 1 or 2 carbon atoms.

A particularly preferred hydrocarbyl group is an alkyl group. As aconsequence, a generalized, but more preferred substituent can berecited by replacing the descriptor “hydrocarbyl” with “alkyl” in any ofthe substituent groups enumerated herein.

Examples of alkyl radicals include methyl, ethyl, n-propyl, isopropyl,n-butyl, isobutyl, sec-butyl, tert-butyl and cyclopropyl. Examples ofsuitable alkenyl radicals include ethenyl (vinyl), 2-propenyl,3-propenyl, 1,4-butadienyl, 1-butenyl, 2-butenyl, and 3-butenyl.Examples of alkynyl radicals include ethynyl, 2-propynyl, 1-propynyl,1-butynyl, 2-butynyl, 3-butynyl, and 1-methyl-2-propynyl.

As a skilled worker will understand, a substituent that cannot existsuch as a C₁ alkenyl group is not intended to be encompassed by the word“hydrocarbyl”, although such substituents with two or more carbon atomsare intended.

Usual chemical suffix nomenclature is followed when using the word“hydrocarbyl” except that the usual practice of removing the terminal“yl” and adding an appropriate suffix is not always followed because ofthe possible similarity of a resulting name to one or more substituents.Thus, a hydrocarbyl ether is referred to as a “hydrocarbyloxy” grouprather than a “hydrocarboxy” group as may possibly be more proper whenfollowing the usual rules of chemical nomenclature. Illustrativehydrocarbyloxy groups include methoxy, ethoxy, n-propoxy, isopropoxy,allyloxy, n-butoxy, iso-butoxy, sec-butoxy, and tert-butoxy groups.

A (C₁-C₇)hydrocarboyl group is a straight, branched chain or cyclic acylhydrocarbyl residue that can contain one to through seven carbon atoms.Illustrative (C₁-C₇)hydrocarboyl groups include formyl, acetyl,propionyl, benzoyl, acryloyl, methacryloyl, cyclopentylcarbonyl,hexanoyl and the like.

Illustrative NR⁴R⁵ substituents where R⁴ and R⁵ are independently thesame or different and are H (hydrido), (C₁-C₆)hydrocarbyl or R⁴ and R⁵together with the depicted nitrogen atom form a 5-7 membered ring thatcan contain one ring oxygen or ring nitrogen atom include amino (NH₂),mono(C₁-C₆)hydrocarbylamino [NH(C₁-C₆)hydrocarbyl],di(C₁-C₆)hydrocarbylamino {N[(C₁-C₆)hydrocarbyl]₂}, as well asN-piperidinyl, N-morphinyl, N-imidazolyl, and N-pyrrolyl substituents.

The term “aryl”, alone or in combination, means an aromatic ring system.Such a ring system includes a phenyl, naphthyl and biphenyl ring system.

A “heteroaryl” group is an aromatic heterocyclic ring that preferablycontains one, or two, or three or four atoms in the ring or rings otherthan carbon. Those heteroatoms can independently be nitrogen, sulfur oroxygen. A heteroaryl group can contain a single 5- or 6-membered ring ora fused ring system having two 6-membered rings or a 5- and a 6-memberedring, or a linked 5,5-, 5,6- or 6,6-membered rings as in a bipyridinylgroup. Exemplary additional heteroaryl groups include 6-membered ringsubstituents such as pyridyl, pyrazyl, pyrimidinyl, and pyridazinyl;5-membered ring substituents such as 1,3,5-, 1,2,4- or 1,2,3-triazinyl,imidazyl, furanyl, thiophenyl, pyrazolyl, oxazolyl, isoxazolyl,thiazolyl, 1,2,3-, 1,2,4-, 1,2,5-, or 1,3,4-oxadiazolyl and isothiazolylgroups; 6-/5-membered fused ring substituents such as benzothiofuranyl,isobenzothiofuranyl, benzisoxazolyl, benzoxazolyl, purinyl andanthranilyl groups; and 6-/6-membered fused rings such as 1,2-, 1,4-,2,3- and 2,1-benzopyronyl, quinolinyl, isoquinolinyl, cinnolinyl,quinazolinyl, and 1,4-benzoxazinyl groups.

The present invention has several benefits and advantages.

One salient benefit is the enhanced potency of the contemplatedN-hydrophobe-substituted compounds (N-hydrophobe-substituted thioamidevancomycins and N-hydrophobe-substituted amidine vancomycins).

Another salient advantage is the greater potency (lessened MIC value)exhibited by a contemplated amidino compound against strains of VanA E.faecalis and E. faecium than against susceptible S. aureus strain.

A further salient benefit is that whereas a hydrophobe-N-substitutedvancomycin and a contemplated, similarly substituted vancomycin amidinecompound have similar potencies against a sensitive S. aureus strain, acontemplated hydrophobe-N-substituted vancomycin amidine compound is, atleast in one instance, about 500 times more potent against than a VanAE. faecalis or E. faecium strain than the correspondinghydrophobe-N-substituted vancomycin.

Yet another salient advantage is that a contemplatedhydrophobe-N-substituted vancomycin thioamide compound exhibits potencyagainst VanA E. faecalis and E. faecium strains that is about the sameas that exhibited by a similarly substituted vancomycin, whereas theunsubstituted vancomycin thioamide was without activity against anybacterial strain examined.

Still further benefits and advantages will be apparent to the skilledworker from the detained description that follows.

DETAILED DESCRIPTION OF THE INVENTION

Contemplated Compound

One aspect of the present invention is a compound or a pharmaceuticallyacceptable salt thereof. A preferred compound of the inventioncorresponds in structure to that shown in Formula I, below,

whereinX═H₂, S or NH; andR is selected from the group consisting of H, (C₁-C₁₆)hydrocarbyl,aryl(C₁-C₆)hydrocarbyldiyl, heteroaryl(C₁-C₆)hydrocarbyldiyl,(C₁-C₆)hydrocarbyldiylheteroaryl, halo-(C₁-C₁₂)-hydrocarbyldiyl, and(C₁-C₁₆)amido substituents, wherein an aryl or heteroaryl group isitself optionally substituted with up to three substituentsindependently selected from the group consisting of:

(i) hydroxy,

(ii) halo,

(iii) nitro,

(iv) (C₁-C₆)hydrocarbyl,

(v) halo(C₁-C₁₆) hydrocarbyl,

(vi) (C₁-C₆)hydrocarbyloxy,

(vii) halo(C₁-C₆)hydrocarbyloxy,

(viii) aryl, and

(ix) aryloxy, wherein an aryl or aryloxy substituent can itself besubstituted with up to three substituents independently selected fromthe group consisting of:

-   -   (i) hydroxy,    -   (ii) halo,    -   (iii) nitro,    -   (iv) (C₁-C₆)hydrocarbyl,    -   (v) halo(C₁-C₁₆)hydrocarbyl,    -   (vi) (C₁-C₆)hydrocarbyloxy, and    -   (vii) halo(C₁-C₆)hydrocarbyloxy; and        R¹ is CH₂OH, CH₂OR², where R² is (C₁-C₇)hydrocarboyl, C(O)OH        [carboxyl], C(O)R³, where R³ is (C₁-C₆)hydrocarbyloxy, or R³ is        NR⁴R⁵, where R⁴ and R⁵ are independently the same or different        and are H (hydrido), (C₁-C₆)hydrocarbyl or R⁴ and R⁵ together        with the depicted nitrogen atom form a 5-7 membered ring that        can contain one ring oxygen atom. In some preferred embodiments,        R is other than hydrido.

When used in a pharmaceutical composition or in a method of treating abacterially-infected mammal in need of antibacterial treatment, the Rgroup of an above compound is other than hydrido (H).

A particularly preferred R substituent is a4-(4′-chlorophenyl)phenylmethyldiyl group, below,

that can also be named a 4-(4′-chlorobiphenyl)methyl group, or a4-(4′-chlorophenyl)benzyl group, and is abbreviated herein as “4-CPB”.

One particularly preferred compound is a thioamido vancomycin compoundthat corresponds in structure to Formula II, below, wherein R and R¹ areas described above.

A still more particularly preferred compound is an amidino vancomycinthat corresponds in structure to Formula III, below, wherein R and R¹are also as described above.

Yet another particularly preferred compound corresponds in structure toFormula IV, below, wherein R and R¹ are also as described above.

Compounds 5, 6, and 17, whose structural formulas are shown below, arethe currently most

preferred compounds contemplated herein. Compound 5 has surprisingactivity as an antibacterial in that it is inactive as the correspondingvancomycin thioamide {[Ψ[C(═S) NH] Tpg⁴]vancomycin}, and is particularlyuseful as an intermediate in the formation of Compound 6. Compound 6 hassurprising antibiotic activity, particularly against VanA E. faecalisand E. faecium.

Compounds 26, 27 and 28, whose structural formulas are shown below, arealso preferred antimicrobial compounds that compounds exhibitantimicrobial activity.

As will be seen from data provided hereinafter, the potency of 4-CPBvancomycin (Compound 4) against a susceptible strain of S. aureus isabout 83 times greater than against VanA strains of E. faecalis and E.faecium. On the other hand, the potency of Compound 6 against those samestrains of VanA E. faecalis and E. faecium was found to be about 6 timesgreater than its potency against the susceptible strain of S. aureus.

Thus, a reversal in potency toward susceptible S. aureus and the VanAstrains of E. faecalis and E. faecium is observed on going from 4-CPBvancomycin to Compound 6 {(4-CPB [Ψ[C(═NH)NH]Tpg⁴]vancomycin}. Thatreversal in potency includes an increase in activity against those VanAstrains of about 500 times on exchanging Compound 6 for 4-CPB vancomycin(Compound 4), with both compounds having about the same potency againstthe susceptible strain of S. aureus.

Another preferred compound that is useful as an intermediate in thesynthesis of Compound 6 and similar compounds is Compound 13, whosestructural formula is shown below.

A compound of the invention can be provided for use by itself, or as apharmaceutically acceptable salt. A contemplated compound of Formula IIis a weak base, whereas an amidine compound of Formula III is a strongerbase, and a compound of Formula IV has a basicity between those ofFormula II and Formula III. A carboxyl group is also present in themolecule that can be present as a carboxylate zwitterion with one of theprotonated amines. That carboxyl group can also be present as part of acarboxylic ester or amide as discussed previously. At physiological pHvalues, a compound of Formula I, such as a compound of Formulas II, IIIor IV is typically present as a salt.

Exemplary salts useful for a contemplated compound include but are notlimited to the following: sulfate, bisulfate, hydrochloride,hydrobromide, acetate, adipate, alginate, citrate, aspartate, benzoate,benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate,digluconate, cyclopentanepropionate, dodecylsulfate, ethanesulfonate,glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate,fumarate, hydrochloride, hydrobromide, hydroiodide,2-hydroxy-ethanesulfonate, lactate, maleate, methanesulfonate,nicotinate, 2-naphthalenesulfonate, oxalate, palmoate, pectinate,persulfate, 3-phenyl-propionate, picrate, pivalate, propionate,succinate, tartrate, thiocyanate, tosylate, mesylate and undecanoate.Salts of the carboxylate group include sodium, potassium, magnesium,calcium, aluminum, ammonium, and the many substituted ammonium salts.

The reader is directed to Berge, J. Pharm. Sci. 1977 68(1):1-19 forlists of commonly used pharmaceutically acceptable acids and bases thatform pharmaceutically acceptable salts with pharmaceutical compounds.

In some cases, a salt can also be used as an aid in the isolation orpurification of a compound of this invention. In such uses, the acidused and the salt prepared need not be pharmaceutically acceptable.

In line with expectations based on the behavior of the correspondingaglycons and in stark contrast to one another, the vancomycin amidinereestablishes potent antimicrobial activity against VanA VRE, whereasvancomycin thioamide is inactive even against vancomycin sensitivebacteria. Introduction of a peripheral 4′-chlorobiphenylmethylmodification into the vancomycin amidine results in a compound with aremarkable spectrum of activity and truly impressive potencies that arelikely derived from cell wall biosynthesis inhibition through twoindependent mechanisms, indicating that such peripheral and pocketsynthetic modifications are synergistic. Such analogs, like vancomycinitself, are likely to display especially durable antibiotic activity[(a) James et al., ACS Chem. Biol. 2012, 7, 797; (b) Boger, Med. Res.Rev. 2001, 21, 356] not prone to rapidly acquired clinical resistance.That 4′-chlorobiphenylmethyl modification into the vancomycin thioamidealso provided some antimicrobial activity to the otherwise bio-inactivecompound.

Composition and Treatment Method

A further aspect of the invention is a method of treating a mammalinfected with a microbial infection such as a bacterial infection,typically a Gram-positive infection; i.e., an infection caused byGram-positive bacteria, and in need of antimicrobial (antibacterial)treatment. In accordance with a contemplated method, anantibacterial-effective amount of one or more compounds of Formula I (acompound of Formulas II, III or IV), such as Compound 6, or apharmaceutically acceptable salt of such a compound is administered toan infected mammal in need.

The compound can be administered as a solid or as a liquid formulation,and is preferably administered via a pharmaceutical compositiondiscussed hereinafter. That administration can also be oral orparenteral, as are also discussed further hereinafter.

It is to be understood that viable mammals are infected with bacteriaand other microbes. The present invention's method of treatment isintended for use against infections of pathogenic microbes that causeillness in the mammal to be treated. Illustrative pathogenic microbesinclude S. aureus, methicilin-resistant S. aureus (MRSA), VanA strainsof E. faecalis and E. feacium, as well as VanB strains of E. faecalis.Evidence of the presence of infection by pathogenic microbes istypically understood by physicians and other skilled medical workers.

A mammal in need of treatment (a subject) and to which a pharmaceuticalcomposition containing a Compound of Formula I or its pharmaceuticallyacceptable salt can be administered can be a primate such as a human, anape such as a chimpanzee or gorilla, a monkey such as a cynomolgusmonkey or a macaque, a laboratory animal such as a rat, mouse or rabbit,a companion animal such as a dog, cat, horse, or a food animal such as acow or steer, sheep, lamb, pig, goat, llama or the like.

As is seen from the data that follow, a contemplated compound is activein in vitro assay studies at less than 1 μg/mL amounts, whichcorresponds to a molar concentration of about 6 to about 60 nanomolar(nM), using the molecular weight of Compound 6. When used in an assaysuch as an in vitro assay, a contemplated compound is typically presentin the composition in an amount that is sufficient to provide aconcentration of about 0.1 nM to about 1 μM to contact microbes to beassayed.

The amount of a compound of Formula I or a pharmaceutically acceptablesalt of such a compound that is administered to a mammal in abefore-discussed method or that is present in a pharmaceuticalcomposition discussed below, which can be used for that administration,is an antibiotic (or antibacterial or antimicrobial) effective amount.It is to be understood that that amount is not an amount that iseffective to kill all of the pathogenic bacteria or other microbespresent in an infected mammal in one administration. Rather, that amountis effective to kill some of the pathogenic organisms present withoutalso killing the mammal to which it is administered, or otherwiseharming the recipient mammal as is well known in the art. As aconsequence, the compound usually has to be administered a plurality oftimes, as is discussed in more detail hereinafter.

A contemplated pharmaceutical composition contains an effectiveantibiotic (or antimicrobial) amount of a Compound of Formula or apharmaceutically acceptable salt thereof dissolved or dispersed in aphysiologically (pharmaceutically) acceptable diluent or carrier. Aneffective antibiotic amount depends on several factors as is well knownin the art. However, based upon the relative potency of a contemplatedcompound relative to that of vancomycin itself for a susceptible strainof S. aureus shown hereinafter, and the relative potencies of vancomycinand a contemplated compound against the VanA E. faecalis and E. faeciumstrains, a skilled worker can readily determine an appropriate dosageamount.

A contemplated composition is typically administered repeatedly in vivoto a mammal in need thereof until the infection is diminished to adesired extent, such as cannot be detected. Thus, the administration toa mammal in need can occur a plurality of times within one day, daily,weekly, monthly or over a period of several months to several years asdirected by the treating physician. More usually, a contemplatedcomposition is administered a plurality of times over a course oftreatment until a desired effect is achieved, typically until thebacterial infection to be treated has ceased to be evident.

A contemplated pharmaceutical composition can be administered orally(perorally) or parenterally, in a formulation containing conventionalnontoxic pharmaceutically acceptable carriers or diluents, adjuvants,and vehicles as desired. The term parenteral as used herein includessubcutaneous injections, intravenous, intramuscular, intrasternalinjection, or infusion techniques. Formulation of drugs is discussed in,for example, Hoover, John E., Remington's Pharmaceutical Sciences, MackPublishing Co., Easton, Pa.; 1975 and Liberman, H. A. and Lachman, L.,Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980.

In some embodiments, a contemplated pharmaceutical composition ispreferably adapted for parenteral administration. Thus, a pharmaceuticalcomposition is preferably in liquid form when administered, and mostpreferably, the liquid is an aqueous liquid, although other liquids arecontemplated as discussed below, and a presently most preferredcomposition is an injectable preparation.

Thus, injectable preparations, for example, sterile injectable aqueousor oleaginous solutions or suspensions can be formulated according tothe known art using suitable dispersing or wetting agents and suspendingagents. The sterile injectable preparation can also be a sterileinjectable solution or suspension in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that can be employed are water,Ringer's solution, and isotonic sodium chloride solution,phosphate-buffered saline.

Other liquid pharmaceutical compositions include, for example, solutionssuitable for parenteral administration. Sterile water solutions of aCompound of Formula I or its salt or sterile solution of a Compound ofFormula I (a compound of Formulas II, III or IV) in a solvent comprisingwater, ethanol, or propylene glycol are examples of liquid compositionssuitable for parenteral administration. In some aspects, a contemplatedCompound of Formula I is provided as a dry powder that is to bedissolved in an appropriate liquid medium such as sodium chloride forinjection prior to use.

In addition, sterile, fixed oils are conventionally employed as asolvent or suspending medium. For this purpose any bland fixed oil canbe employed including synthetic mono- or diglycerides. In addition,fatty acids such as oleic acid find use in the preparation of aninjectable composition. Dimethyl acetamide, surfactants including ionicand non-ionic detergents, polyethylene glycols can be used. Mixtures ofsolvents and wetting agents such as those discussed above are alsouseful.

A sterile solution can be prepared by dissolving the active component inthe desired solvent system, and then passing the resulting solutionthrough a membrane filter to sterilize it or, alternatively, bydissolving the sterile compound in a previously sterilized solvent understerile conditions.

Solid dosage forms for oral administration can include capsules,tablets, pills, powders, and granules. The amount of a contemplatedCompound or salt of Formula I such as Compound 6 in a solid dosage formis as discussed previously, an amount sufficient to provide an effectiveantibiotic (or antimicrobial) amount. A solid dosage form can also beadministered a plurality of times during a one week time period.

In such solid dosage forms, a compound of this invention is ordinarilyadmixed as a solution or suspension in one or more diluents appropriateto the indicated route of administration. If administered per os, thecompounds can be admixed with lactose, sucrose, starch powder, celluloseesters of alkanoic acids, cellulose alkyl esters, talc, stearic acid,magnesium stearate, magnesium oxide, sodium and calcium salts ofphosphoric and sulfuric acids, gelatin, acacia gum, sodium alginate,polyvinylpyrrolidone, and/or polyvinyl alcohol, and then tableted orencapsulated for convenient administration. Such capsules or tablets cancontain a controlled-release formulation as can be provided in adispersion of active compound in hydroxypropylmethyl cellulose. In thecase of capsules, tablets, and pills, the dosage forms can also comprisebuffering agents such as sodium citrate, magnesium or calcium carbonateor bicarbonate. Tablets and pills can additionally be prepared withenteric coatings.

A mammal in need of treatment (a subject) and to which a pharmaceuticalcomposition containing a Compound of Formula I or a pharmaceuticallyacceptable salt thereof is administered can be a primate such as ahuman, an ape such as a chimpanzee or gorilla, a monkey such as acynomolgus monkey or a macaque, a laboratory animal such as a rat, mouseor rabbit, a companion animal such as a dog, cat, horse, or a foodanimal such as a cow or steer, sheep, lamb, pig, goat, llama or thelike.

Where an in vitro assay is contemplated, a sample to be assayed such ascells and tissue can be used. These in vitro compositions typicallycontain water, sodium or potassium chloride, and one or more buffersalts such as and acetate and phosphate salts, Hepes or the like, ametal ion chelator such as EDTA that are buffered to a desired pH valuesuch as pH 4.0-8.5, preferably about pH 7.2-7.4, depending on the assayto be performed, as is well known.

Preferably, the pharmaceutical composition is in unit dosage form. Insuch form, the composition is divided into unit doses containingappropriate quantities of the active compound. The unit dosage form canbe a packaged preparation, the package containing discrete quantities ofthe preparation, for example, in vials or ampules.

Antimicrobial Activity

The pocket-modified vancomycin analogues that contain the C-terminushydroxymethyl group (Compounds 23-25), their chlorobiphenyl derivatives(Compounds 26-28), as well as the fully functionalized vancomycinanalogues [Ψ[C(═S)NH]Tpg⁴]vancomycin, [Ψ[C(═NH)NH]Tpg⁴]-vancomycin, and[Ψ[CH₂NH]Tpg⁴]vancomycin (Compounds 2, 3 and 16) and their(4-chlorobiphenyl)methyl derivatives (Compounds 5, 6 and 17) wereexamined alongside the corresponding vancomycin (residue 4 amide)derivatives. The antimicrobial activity of the compounds was evaluatedagainst a panel of Gram-positive bacteria that includevancomycin-sensitive S. aureus (VSSA), methicillin-resistant S. aureus(MRSA), and both VanA (E. faecalis and E. faecium) and VanB (E.faecalis) vancomycin-resistant Enterococci (VRE) of which VanA is themost stringent of the resistant organisms, with those results shown inthe tables below.

Notably, one VanA VRE (E. faecium, ATCC BAA-2317) tested represents anemerging challenging multidrug resistant VanA VRE that is not onlyresistant to vancomycin and teicoplanin, but also ampicillin,benzylpenicillin, ciprofloxacin, erythromycin, levofloxacin,nitrofurantoin, tetracycline. It is also insensitive to linezolid, butremains sensitive to tigecycline and dalfopristine.

Antimicrobial Activity, MIC^(a) (μg/mL)

sensitive MRSA VanA VanB S. aureus ^(b) S. aureus ^(c) E. faecalis ^(d)E. faecium ^(e) E. faecalis ^(f) R = H  1, X = O 0.5 0.5 250 250 8  2, X= S >32 >32 >32 >32 >32  3, X = NH nd^(g) nd^(g) 0.5 0.5 nd^(g) 16, X =H₂ nd^(g) nd^(g) 31 31 nd^(g) R = CBP, (4-chlorobiphenyl)methyl  4, X =O 0.03 0.03 2.5 2.5 0.03  5, X = S 2 2 4 4 2  6, X = NH 0.03 0.06 0.0050.005 0.06 17, X = H₂ 0.5 0.25 0.13 0.06 0.5 ^(a)MIC = Minimuminhibitory concentration. ^(b)ATCC 25923. ^(c)ATCC 43300. ^(d)BM 4166.^(e)ATCC BAA-2317. ^(f)ATCC 51299. ^(g)not determined.

As is seen from the data in the table below, the activity of C-terminushydroxymethyl derivatives paralleled that observed with thecorresponding C-terminus carboxylic acids. However, the C-terminushydroxymethyl compounds displayed the same general trends and nearidentical absolute MIC values, reinforcing the generality andsignificance of the conclusions.

Antimicrobial Activity, MIC^(a) (μg/mL)

sensitive MRSA VanA VanB S. aureus ^(b) S. aureus ^(c) E. faecalis ^(d)E. faecium ^(e) E. faecalis ^(f) R = H 23, X = O 0.5 0.5 250 250 8 24, X= S >32 >32 >32 >32 >32 25, X = NH nd^(g) nd^(g) 2 2 nd^(g) R = CBP,(4-chlorobiphenyl)methyl 26, X = O 0.03 0.03 2 4 0.13 27, X = S 4 8 8 44 28, X = NH 0.13 0.13 0.02 0.02 0.06 ^(a)MIC = Minimum inhibitoryconcentration. ^(b)ATCC 25923. ^(c)ATCC 43300. ^(d)BM 4166. ^(e)ATCCBAA-2317. ^(f)ATCC 51299. ^(g)not determined.

Vancomycin-sensitive S. aureus (VSSA, ATCC 25923): sensitive tovancomycin, teicoplanin, oritavancin, daptomycin, linezolid,quinupristin-dalfopristin, fussidic acid, azithromycin, telithromycin,gentamycin, penicillin V, nafcillin, ampicillin, oxacillin,ciprofloxacin, levofloxacin, garenoxacin and moxifloxacin.Methicillin-resistant S. aureus (MRSA, ATCC 43300): sensitive tovancomycin, teicoplanin, daptomycin, linezolid, tigecycline andciprofloxacin; resistant to methicillin, amoxicillin, amoxicillin withclavulanic acid, cephalexin, enrofloxacin, erythromycin, azithromycin,gentamycin, clindamycin, lincomycin-spectinomycin, neomycin, oxacillin,penicillin G, streptomycin, trimethoprim-sulfamethoxazole andtetracycline. VanA E. faecalis (VanA VRE, BM 4166): resistant toerythromycin, gentamicin, chloramphenicol, and ciprofloxacin as well asvancomycin and teicoplanin; sensitive to daptomycin. VanA E. faecium(VanA VRE, ATCC BAA-2317): resistant to ampicillin, benzylpenicillin,ciprofloxacin, erythromycin, levofloxacin, nitrofurantoin, andtetracycline as well as vancomycin and teicoplanin, insensitive tolinezolid; sensitive to tigecycline and dalfopristine. VanB E. faecalis(VanB VRE, ATCC 51299): resistant to vancomycin, streptomycin,gentamicin; sensitive to teicoplanin, ampicillin, tetracycline, andciprofloxacin.

The activity of the pocket modified vancomycin analogues Compounds 2, 3and 16 matched the in vitro antimicrobial activity of the correspondingaglycon analogue Compounds 8, 9 and 10 on which they are based. Althoughit is well established that the attached unmodified carbohydrate doesnot alter in vitro antimicrobial activity (potency) or influence targetD-Ala-D-Ala or D-Ala-D-Lac binding, the vancomycin disaccharide impactsit's in vivo activity; increasing water solubility, influencingpharmacokinetic and distribution properties, and contributing apotential second mechanism of action.

An analogous impact on the vancomycin analogue Compounds 2, 3 and 16might be expected because each represents the change of a single atom inthe binding pocket (residue 4 carbonyl O→S, NH, H₂), and they would bethe preferred compounds (vs Compounds 8, 9 and 10) with which to probein vivo activity.

Within this series, vancomycin displayed potent activity against VSSAand MRSA (MIC=0.5 μg/mL), but was ineffective against VanA VRE (MIC=250μg/mL) and only modestly active against VanB VRE (MIC=8 μg/mL) under theassay conditions employed. Consistent with its lack of binding to eitherD-Ala-D-Ala or D-Ala-D-Lac, the thioamide Compound 2 proved inactive asan antimicrobial agent (MICs>32 μg/mL) against both sensitive andresistant bacteria.

Both the amidine Compound 3 [Okano et al., J. Am. Chem. Soc. 2014, 136,13522] and the methylene analogue Compound 16 reinstated activityagainst VanA VRE (BM4166) with MICs of 0.5 and 31 μg/mL, respectively.This finding was precisely in line with expectations based on therelative dual D-Ala-D-Ala and D-Ala-D-Lac binding affinities of theaglycons and matching the activities observed with the correspondingaglycons Compounds 9 and 10.

Of most significance, the amidine Compound 3 displayed a potency againstVanA VRE that matched the activity vancomycin displays against sensitivebacteria (VSSA and MRSA, MICs=0.5 μg/mL).

Given the distinct origins of their impact on the antimicrobial activityof vancomycin, it was expected that incorporation of the peripheralchlorobiphenyl modification into the structure of the bindingpocket-modified vancomycin analogues would further increase theirantimicrobial activity against not only sensitive, but alsovancomycin-resistant bacteria to truly remarkable potencies. Althoughthis conceivably could have been demonstrated by substitution of thesynthetic aglycon Compounds 7, 8, 9 and 10, the most definitiveassessment of the dual impact was expected to be a direct comparison ofchlorobiphenyl vancomycin (Compound 4) with Compounds 5, 6, and 17,wherein a series of key changes in a single atom in the binding pocketwere introduced, despite the synthetic challenges this posed. Thischoice of both the site of modification and the use of thechlorobiphenyl modification proved important to understanding thebehavior of such analogues and revealed unique insights into the originof the effects.

In line with reports of its impact, introduction of the(4-chlorobiphenyl)methyl group into vancomycin (Compound 4 vsCompound 1) results in 100-fold improvements in the activity againstVanA and VanB VRE (MIC=2.5 vs 250 μg/mL) and 20-fold improvementsagainst VSSA and MRSA (MIC=0.03 vs 0.5 μg/mL) in the strains examined.In spite of the increases in potency, it remains 100-fold less effectiveagainst VanA VRE.

Both the amidine Compound 6 and the methylene analogue Compound 17exhibited the same 100-fold increases in activity against VanA VRE,exhibiting remarkable MICs of 0.005 μg/mL and 0.06-0.13 μg/mL,respectively. Just as significantly, introduction of the chlorobiphenylgroup into either the vancomycin amidine Compound 6 or the vancomycinmethylene analogue Compound 17 resulted in compounds with remarkablespectra of activity at these impressive potencies.

Both compounds were equally effective against both vancomycin-sensitivebacteria (VSSA and MRSA) and vancomycin-resistant bacteria (VanA andVanB VRE) of which VanA VRE proved especially sensitive to theanalogues. Both analogues exhibit MICs below 1 μg/mL across thebacterial panel, and the amidine Compound 6 was found to be on average15-fold more potent than the methylene analogue Compound 17, preciselyin line with their relative dual ligand binding affinities.

Moreover, the amidine Compound 6 not only matches the activity thatCBP-vancomycin (Compound 4) displays against vancomycin-sensitivebacteria (VSSA and MRSA), but it also exhibits this extraordinarypotency against VanA and VanB vancomycin-resistant bacteria. In fact,the activity of Compound 6 against the most stringent of the resistantbacteria, VanA VRE, was nearly 10-fold better than the potency itdisplays against the sensitive bacteria, representing a 500-foldincrease in activity relative to CBP-vancomycin (Compound 4) and a50,000-fold increase in activity relative to vancomycin (Compound 1)itself. Thus, the chlorobiphenyl introduction into the pocket modifiedvancomycin analogues Compound 6 (MICs=0.005-0.06 μg/mL) and Compound 17(MICs=0.06-0.5 μg/mL) synergistically increased their potency againstboth vancomycin-sensitive and vancomycin-resistant bacteria.

Insights into this behavior came from the examination of thechlorobiphenyl derivative of the vancomycin thioamide (Compound 5).Introduction of the (4-chlorobiphenyl)methyl group into vancomycinthioamide (Compound 2) with Compound 7 reinstates impressive and equallypotent activity (MIC=2-4 μg/mL) against all vancomycin-sensitive andvancomycin-resistant strains despite its inability to bind the primarycell wall target D-Ala-D-Ala/D-Ala-D-Lac.

It is unlikely such effective activity can be achieved simply by theeffects of antibiotic membrane anchoring, antibiotic dimerization, ordisruption of bacterial membrane integrity. Rather, it likely reflectspotent antimicrobial activity derived from a second mechanism of actionimpacting cell wall synthesis unrelated to D-Ala-D-Ala/D-Ala-D-Lacbinding.

In line with observations made with CBP-vancomycin and analoguescontaining damaged binding pockets, this most likely involves potenttransglycosylase inhibition mediated by direct binding to the enzyme[(a) Ge et al., Science 1999, 284, 507; (b) Chen et al., Proc. Natl.Acad. Sci. USA 2003, 100, 5658; and (c) Goldman et al., FEMS Microbiol.Lett. 2000, 183, 209]. Because of the insights derived from thecomparative examination of the thioamide Compounds 2 and 5, the behaviorof the CBP-vancomycin amidine Compound 6 and CBP-vancomycin methyleneanalogue Compound 17 likely represents a spectrum of activity andpotency derived from bacterial cell wall synthesis inhibition throughtwo synergistic mechanisms, one involving inhibition oftranspeptidase-catalyzed cell wall cross-linking through dual substrate(D-Ala-D-Ala and D-Ala-D-Lac) binding and the second through directinhibition of transglycosylase independent of such ligand binding.

If this is the case, it suggests that resistance is unlikely to emergeagainst such analogues because it would entail simultaneous bacterialchanges to two distinct targets of the antibiotics, one of which is notsubject to direct genetic alterations. As such, both Compound 6 andCompound 17 are superb candidates for preclinical development. Theirpreliminary assessments not only indicate that they address the presentday emerging vancomycin resistance and exhibit remarkable spectrums ofactivity and superb antimicrobial potency, but also that they areendowed with a unique combination of characteristics that may allow themto display the 50 year clinical durability of vancomycin.

Although at this stage still speculative, the four chlorobiphenylderivative Compounds 4, 5, 6 and 17 are also uniquely poised to helpunravel the subtleties of the mechanisms of action of such modifiedglycopeptide antibiotics. Due to its inability to bind eitherD-Ala-D-Ala or D-Ala-D-Lac, the thioamide Compound 5(CBP-[Ψ[C(═S)NH]Tpg⁴]-vancomycin) derives its antimicrobial activity(MIC=2-4 μg/mL) exclusively through a distinct second mechanism ofaction that does not involve ligand binding and likely involves directinhibition of transglycosylase [(a) Ge et al., Science 1999, 284, 507;(b) Chen et al., Proc. Natl. Acad. Sci. USA 2003, 100, 5658; and (c)Goldman et al., FEMS Microbiol. Lett. 2000, 183, 209].

By virtue of its inability to bind D-Ala-D-Lac, CBP-vancomycin (Compound4) also likely derives its similar activity against vancomycin-resistantorganisms (VanA VRE, MIC=2.5 μg/mL) by this same mechanism potentiallyinvolving only the direct inhibition of transglycosylase, whereas itsmore potent activity against vancomycin-sensitive organisms (VSSA andMRSA, MIC=0.03 μg/mL) is derived from the equally potent and synergisticinhibition of both transpeptidase (via D-Ala-D-Ala binding and substratesequestration) and transglycosylase (direct enzyme inhibition).

As a result of the binding pocket redesign and ability to exhibit fullyeffective dual D-Ala-D-Ala and D-Ala-D-Lac binding combined with theperipheral chlorobiphenyl-mediated potential direct inhibition oftransglycosylase, Compound 6 {CBP-[Ψ[C(═NH)NH]Tpg⁴]vancomycin} picks upthe ability to effectively inhibit transpeptidase invancomycin-resistant bacteria (VanA VRE, via D-Ala-D-Lac binding),maintains the ability to inhibit transpeptidase in vancomycin-sensitivebacteria (VSSA and MRSA, via D-Ala-D-Ala binding), permits the potentialindirect transglycosylase inhibition through ligand binding, andbenefits potentially from an equally potent and synergistic directinhibition of transglycosylase independent of D-Ala-D-Ala or D-Ala-D-Lacbinding. The net result is an antibiotic that benefits from two equallypotent, independent, and synergistic mechanisms of action and thatdisplays the remarkable antimicrobial potencies (MIC=0.06-0.005 μg/mL)against both vancomycin-sensitive and vancomycin-resistant bacteria.

In contrast but similarly interestingly, the potency ofCBP-[Ψ[CH₂NH]Tpg⁴]vancomycin (Compound 17) (MIC=0.5-0.06 μg/mL) suggeststhat the principle mechanism by which it acts is through the potentialchlorobiphenyl-mediated direct inhibition of transglycosylase, but nowwith a second less potent contribution derived from its balanced, albeitreduced, dual ligand binding affinities for inhibition of transpeptidasein either vancomycin-sensitive and vancomycin-resistant bacteria. It isremarkable that the series appears to display the trends of twoindependent mechanisms, which act synergistically with one another, toprovide newly predictable potency trends derived independently from thebinding pocket modifications and the peripheral carbohydratesubstitution.

Kahne and co-workers have shown that although the potency of mostlipid-linked glycopeptides or their aglycons lose activity against VanAstrains when their binding pocket is chemically damaged [(a) Chen etal., Tetrahedron 2002, 58, 6585; and (b) Kerns et al., J. Am. Chem. Soc.2000, 122, 12608-12609], indicating ligand binding is important to theiractivity, a small subset including CBP-vancomycin retains goodantimicrobial activity even when their binding pocket is chemicallydamaged [(a) Ge et al., Science 1999, 284, 507; and (b) Chen et al.,Proc. Natl. Acad. Sci. USA 2003, 100, 5658]. Moreover, it is suchderivatives that were shown by Kahne and Walker [Chen et al., Proc.Natl. Acad. Sci. USA 2003, 100, 5658] to effectively inhibittransglycosylase without substrate or ligand binding, suggesting itdirectly binds and inhibits the enzyme. CBP-[Ψ[C(═S)NH]Tpg⁴]vancomycinembodies these same characteristics, displays the same VanA VRE potency,and likely will display the same behavior toward transglycosylase. It islikely that this activity against VanA strains requires a specificpositioning of the hydrophobic substituent attached to the vancomycindisaccharide. As a consequence, it is especially notable that thesestudies were conducted with single atom changes to the binding pocket ofvancomycin and CBP-vancomycin and not conducted on simpler, moreaccessible aglycon derivatives.

The activity of many antibiotics, especially cationic peptideantibiotics, display changes in activity with additives [Moeck,Antimicrob Agents Chemother 2008, 5, 159] including oritavancin, or canbe dependent on the broth conditions [Otvos et al., In Methods inMolecular Biol., 2007, Vol 386, Fields ed.; Humana Press, Totowa, N.J.,pp 309-320]. The vancomycin, [Ψ[C(═S)NH]Tpg⁴]-vancomycin, and[Ψ[CH₂NH]Tpg⁴]vancomycin derivatives exhibited small 2-4 fold shifts inantimicrobial activity with variations in the broth dilution, whereas[Ψ[C(═NH)NH]Tpg⁴] vancomycin varied more.

Antimicrobial Activity, MIC^(a) (μg/mL)

sensitive MRSA Van A Van B S. aureus ^(b) S. aureus ^(c) E. faecalis^(d) E. faecium ^(e) E. faecalis ^(f) Serum concentration 10% 25% 100%10% 25% 100% 10% 25% 100% 10% 25% 100% 10% 25% 100% R = H  1, X = O 0.50.5 0.5 0.5 0.5 1 250 250 500 250 250 500 4 8 32 33, X =S >32 >32 >32 >32 >32 >32 >32 >32 >32 >32 >32 >32 >32 >32 >32 34, X = NHnd^(g) nd^(g) nd^(g) nd^(g) nd^(g) nd^(g) 0.5 8 >32 0.5 4 >32 nd^(g)nd^(g) nd^(g) 35, X = CH₂ nd^(g) nd^(g) nd^(g) nd^(g) nd^(g) nd^(g) 3162 125 31 62 125 nd^(g) nd^(g) nd^(g) R = CBP, (4-chlorobiphenyl)methyl36, X = O 0.03 0.03 0.06 0.03 0.06 0.06 2.5 5 10 2.5 5 5 0.03 0.06 0.0637, X = S 2 2 4 2 4 4 4 4 8 4 4 8 2 2 8 38, X = NH 0.03 0.12 1 0.06 0.241 0.005 0.06 0.5 0.005 0.06 0.5 0.06 0.12 1 39, X = CH₂ 0.5 1 2 0.25 0.51 0.13 0.26 0.5 0.06 0.13 0.5 0.5 1 2 ^(a)MIC = Minimum inhibitoryconcentration. ^(b)ATCC 25923. ^(c)ATCC 43300. ^(d)BM 4166. ^(e)ATCCBAA-2317. ^(f)ATCC 51299. ^(g)not determined.Synthetic Procedures

Compounds 23-28 were prepared as described hereinafter and in (a) Xie etal., J. Am. Chem. Soc. 2011, 133, 13946; and (b) Xie et al., J. Am.Chem. Soc. 2012, 134, 1284. The general synthesis strategy is laid outin Scheme 1 hereinafter.

The synthesis of Compound 3 was accomplished by enlisting two sequentialenzymatic glycosylation reactions to first provide[Ψ[C(═S)NH]Tpg⁴]vancomycin (Compound 2), followed by a finalAg(I)-promoted [Okano et al., J. Am. Chem. Soc. 2012, 134, 8790]conversion of the residue 4 thioamide to an amidine. Compound 2 wasconverted to Compound 5 by an additional single-step introduction of theN-4-(4′-chlorobiphenyl)methyl group into [Ψ[C(═S)NH]Tpg⁴]vancomycin,that step was followed by Ag(I)-promoted conversion of the thioamide toan amidine to afford Compound 6.

In addition to providing the opportunity to assess[Ψ[C(═NH)NH]Tpg⁴]vancomycin (Compound 3) and the impact of combining thevancomycin pocket redesign with a key peripheral structuralmodification, the approach was designed to shed light on the role of thechlorobiphenyl modification with the examination of[Ψ[C(═S)NH]Tpg⁴]vancomycin (Compound 2) and its4-(4′-chlorobiphenyl)methyl derivative, which are incapable of bindingD-Ala-D-Ala or D-Ala-D-Lac.

The recombinant glycosyltranferases GtfE and GtfD from the vancomycinproducing strain of A. orientalis (ATCC 19795) were expressed in E. colifrom the corresponding constructs [(a) Losey et al., Biochemistry 2001,40, 4745; (b) Oberthur et al., J. Am. Chem. Soc. 2005, 127, 10747; (c)Losey et al., Chem. Biol. 2002, 9, 1305; (d) Thayer et al., Chem. AsianJ. 2006, 1, 445] (a gift of C. Walsh) and were purified to homogeneity(His₆ tag). The two sequential glycosylations of synthetic Compound 8[(a) Xie et al., J. Am. Chem. Soc. 2011, 133, 13946; (b) Xie et al., J.Am. Chem. Soc. 2012, 134, 1284] were conducted with the purifiedglycosyltransferases enzymes and the synthetic glycosyl donors(UDP-glucose [Sigma-Aldrich] for GtfE and UDP-vancosamine [(a) Nakayamaet al., Org. Lett. 2014, 16, 3572; (b) Oberthur et al., Org. Lett. 2004,6, 2873] for GtfD) under recently described conditions [(a) Nakayama etal., Org. Lett. 2014, 16, 3572; (b) Oberthur et al., Org. Lett. 2004, 6,2873] to provide the pseudoaglycon Compound 13 (75%) and[Ψ[C(═S)NH]Tpg⁴]-vancomycin (Compound 2, 52%) (Scheme 1).

Direct conversion of the thioamide to the corresponding amidine (10equiv AgOAc, sat. NH₃-MeOH, 25° C., 7 hours) [Okano et al., J. Am. Chem.Soc. 2012, 134, 8790] provided [Ψ[C(═NH)NH]Tpg⁴]vancomycin (Compound 3).Significantly, the reaction was capable of implementation withoutcompetitive deglycosylation and the entire sequence (conversion ofCompound 8 to Compound 3) was conducted without the use of intermediateprotecting groups.

Subsequent introduction of the 4′-chlorobiphenylmethyl group into[Ψ[C(═S)NH]Tpg⁴]-vancomycin (Compound 2) by selective reductiveamination (1.5 equiv 4-(4′-chlorophenyl)benzaldehyde, 5 equiv i-Pr₂NEt,DMF, 70° C., 2 hours; NaCNBH₃, 70° C., 5 hours) provided Compound 5(57%) without observation of competitive reactions of either thethioamide (reduction) or the N-terminal free amine (reductiveamination), using conditions modified from those disclosed forchlorobiphenyl vancomycin itself [Chen et al., Tetrahedron 2002, 58,6585].

Direct AgOAc-promoted (10 equiv. sat. NH₃-MeOH, 25° C., 7 hours)conversion of the thioamide to the amidine provided Compound 6 (45%,unoptimized), the chlorobiphenylmethyl derivative of[Ψ[C(═NH)NH]Tpg⁴]vancomycin (Compound 3), without the need forintervening protecting groups throughout the 4-step sequence. By design,the final reaction introducing the amidine functionality was conductedeffectively on fully functionalized substrates (Compound 2 and Compound5), lacking protecting groups and incorporating the vancomycindisaccharide.

More particularly, for vancomycin, the carbohydrate introduction hasbeen approached by using either chemical [(a) Ge et al., J. Am. Chem.Soc. 1998, 120, 11014; (b) Thompson et al., J. Am. Chem. Soc. 1999, 121,1237; (c) Leimkuhler et al., Tetrahedron: Asymmetry 2005, 16, 599; (d)Nicolaou et al., Angew. Chem. Int. Ed. 1999, 38, 240; (e) Nicolaou etal., E. Chem. Eur. J. 1999, 5, 2648; (f) Ritter et al., Angew. Chem.Int. Ed. 2003, 42, 4657] or enzymatic [(a) Losey et al., Biochemistry2001, 40, 4745; (b) Oberthur et al., J. Am. Chem. Soc. 2005, 127, 10747;(c) Losey et al., Chem. Biol. 2002, 9, 1305; (d) Dong et al., J. Am.Chem. Soc. 2002, 124, 9064; (e) Kruger et al., Chem. Biol. 2005, 12,131; (f) Thayer et al., Chem. Asian J. 2006, 1, 445; (g) Thayer et al.,Angew. Chem. Int. Ed. 2005, 44, 4596; (h) Solenberg et al., Chem. Biol.1997, 4, 195; (i) Fu et al., Org. Lett. 2005, 7, 1513; and (j) Fu etal., Nat. Biotechnol. 2003, 21, 1467] glycosylations for sequentialintroduction of the glucose and vancosamine sugars located on thecentral residue of the aglycon or pseudoaglycon, respectively.

Of these and as noted elsewhere [(a) Ge et al., J. Am. Chem. Soc. 1998,120, 11014; (b) Thompson et al., J. Am. Chem. Soc. 1999, 121, 1237; (c)Leimkuhler et al., Tetrahedron: Asymmetry 2005, 16, 599; (d) Losey etal., Biochemistry 2001, 40, 4745; (e) Oberthur et al., J. Am. Chem. Soc.2005, 127, 10747; (f) Losey et al., Chem. Biol. 2002, 9, 1305; (g) Donget al., J. Am. Chem. Soc. 2002, 124, 9064; (h) Kruger et al., Chem.Biol. 2005, 12, 131; (i) Thayer et al., Chem. Asian J. 2006, 1, 445; and(j) Thayer et al., Angew. Chem. Int. Ed. 2005, 44, 4596] the enzymaticglycosylations avoid protection and the corresponding deprotection ofaglycon precursors required of chemical procedures, providing the fullyglycosylated products in 2-steps from the fully deprotected aglycons. Asa consequence, the sequential glycosylations of the modified aglyconderivatives were examined alongside the vancomycin aglycon and itsC-terminus hydroxymethyl derivative using the enzymatic approach[Nakayama et al., Org. Lett. 2014, 16, 3572].

The recombinant glycosyltranferases GtfE and GtfD from the vancomycinproducing strain of A. orientalis (ATCC 19795) were expressed in E. colifrom the corresponding constructs [Losey et al., Biochemistry 2001, 40,4745] and were purified to homogeneity (His₆ tag). Notably and althoughthe endogenous glycosyl donors for both enzymes are the TDP-sugars [(a)Losey et al., Biochemistry 2001, 40, 4745; (b) Oberthur et al., J. Am.Chem. Soc. 2005, 127, 10747; (c) Losey et al., Chem. Biol. 2002, 9,1305; (d) Dong et al., J. Am. Chem. Soc. 2002, 124, 9064; (e) Kruger etal., Chem. Biol. 2005, 12, 131], UDP-sugars have been shown to be aseffective co-substrates for both enzymes. Because the requisiteNDP-sugar precursor UMP morpholidate is commercially available from fourcommercial suppliers, including Sigma-Aldrich, whose product was usedherein and the corresponding activated TMP is not, UDP-vancosamine wasused with GtfD [Nakayama et al., Org. Lett. 2014, 16, 3572].

The UDP-vancosamine, possessing the required β-anomer stereochemistry,was prepared by a procedure described in Oberthur et al., Org. Lett.2004, 6, 2873 to access TDP-vancosamine with modifications to thesynthetic route that incorporate uridine versus thymidine [Nakayama etal., Org. Lett. 2014, 16, 3572]. With use of the purified enzymes andthe synthetic glycosyl donors UDP-glucose (also from Sigma-Aldrich forGtfE) and UDP-vancosamine [Nakayama et al., Org. Lett. 2014, 16, 3572](for GtfD), conditions were optimized for the two sequentialglycosylations of vancomycin aglycon (Compound 7) as well as itsC-terminus hydroxymethyl derivative [Nakayama et al., Org. Lett. 2014,16, 3572].

Of the two glycosylation reactions, the initial GtfE-catalyzedincorporation of glucose using UDP-glucose exhibited the greatestaglycon substrate sensitivity and those bearing a C-terminushydroxymethyl group were established to be much less effective than thecorresponding carboxylic acids. Previously reported optimization effortsfocused on this glycosylation reaction and examined along with both thevancomycin aglycon (Compound 7) and the corresponding hydroxymethylsubstrate (37° C.).

In the case of the hydroxymethyl substrate, whose reaction proceeded ata slow rate, preparative amounts of product pseudoaglycon (55%, 48hours) [Nakayama et al., Org. Lett. 2014, 16, 3572] were obtained byincreasing the amount of enzyme used (20 vs 5 μM). The residue 4thioamide (Compound H) and the residue 4 methylene (Compound I)derivatives were capable of glycosylation using GtfE and UDP-glucose toprovide the pseudoaglycons Compound 20 (35%; 65% based on recoveredstarting material, 25 μM GtfE) and Compound 22 (HPLC scale, 22% with 5μM GtfE), whereas glycosylation of the residue 4 amidine was notsufficient to provide isolatable amounts of product.

Whereas the studies with the C-terminal hydroxymethyl amide andthioamide were conducted on preparative scales, the studies with theC-terminal hydroxymethyl amidine and the more recent methylenederivative were only conducted on an analytical scale as a prelude tostudies with the corresponding and more effective C-terminus carboxylicacids.

The second glycosylation reaction catalyzed by GtfD using syntheticUDP-vancosamine proceeded to completion rapidly (<3 hours) independentof the substrate, displaying no impact of either the C-terminushydroxymethyl substituent or nature of residue 4 (amide, thioamide, ormethylene), and the reaction conditions required little optimization.Aside from incorporating glycerol (10% v/v) and reducing the amount ofadded BSA (0.2 vs 1 mg/mL), the conditions used are essentially thoseoriginally disclosed [(a) Losey et al., Biochemistry 2001, 40, 4745; (b)Oberthur et al., Am. Chem. Soc. 2005, 127, 10747; (c) Losey et al.,Chem. Biol. 2002, 9, 1305; (d) Dong et al., J. Am. Chem. Soc. 2002, 124,9064; and (e) Kruger et al., Chem. Biol. 2005, 12, 131] for use of thisenzyme and provided both Compound 23 (79%) [Nakayama et al., Org. Lett.2014, 16, 3572] and Compound 24 (84%) in excellent yields.

Direct conversion of thioamide Compound 24 to the corresponding amidine[10 equiv AgOAc (Corey et al., Tetrahedron Lett. 1978, 5) sat. NH₃-MeOH,25° C., 6 hours, 50%] (Okano et al., J. Am. Chem. Soc. 2012, 134, 8790)provided Compound 25, the C-terminus hydroxymethyl analogue ofvancomycin containing the residue 4 amidine modification. Importantly,this latter reaction was implemented without competitive deglycosylationand the entire 3-step sequence could be conducted without protectinggroups. Most significantly, the approach defined an effective route tothe key residue 4 amidine analogues despite their failure to directlyparticipate effectively in the initial enzymatic glycosylation reaction.

Subsequent introduction of the chlorobiphenyl group into Compounds 23and 24 by selective reductive amination was conducted best withpreformation of the imine (1.3-1.5 equiv4-(4′-chlorophenyl)benzaldehyde, 5 equiv i-Pr₂NEt, DMF, 30° C., 9-12hours) followed by subsequent imine reduction [100 equiv NaBH(OAc)₃, 30°C., 2 hours] and provided Compound 26 (67-74%) and Compound 27 (74%)using conditions modified [NaBH(OAc)₃ vs NaCNBH₃] from those disclosedfor (4-chlorobiphenyl)methyl vancomycin itself [(a) Chen et al.,Tetrahedron 2002, 58, 6585; (b) Kerns et al., J. Am. Chem. Soc. 2000,122, 12608-12609].

Of most significance, the reaction of the latter compound occurs withoutobservation of competitive reactions of either the residue 4 thioamide(reduction) or the N-terminal free amine (reductive amination). A finalAgOAc-promoted (10 equiv, sat. NH₃-MeOH, 22° C., 6 hours) conversion ofthe thioamide Compound 27 to the amidine provided Compound 28 (48%,unoptimized), the 4′-chlorobiphenyl derivative of Compound 25. Bydesign, the final reaction introducing the amidine as well as thesequential glycosylation reactions and the reductive amination could beconducted effectively on fully functionalized substrates, lackingprotecting groups and incorporating the vancomycin disaccharide.

Subsequent introduction of the 4′-chlorobiphenyl group into Compounds 23and 24 by selective reductive amination was conducted best withpreformation of the imine (1.3-1.5 equiv4-(4′-chlorophenyl)benzaldehyde, 5 equiv i-Pr₂NEt, DMF, 30° C., 9-12hours) followed by subsequent imine reduction (100 equiv NaBH(OAc)₃, 30°C., 2 hours) and provided Compound 26 (67-74%) and Compound 27 (74%)using conditions modified (NaBH(OAc)₃ vs NaCNBH₃) from those disclosedfor (4′-chlorobiphenyl)methyl vancomycin itself [(a) Chen et al.,Tetrahedron 2002, 58, 6585; (b) Kerns et al., J. Am. Chem. Soc. 2000,122, 12608-12609].

Of most significance, the reaction of the latter compound occurs withoutobservation of competitive reactions of either the residue 4 thioamide(reduction) or the N-terminal free amine (reductive amination). A finalAgOAc-promoted (10 equiv, sat. NH₃-MeOH, 22° C., 6 hours) conversion ofthe thioamide Compound 27 to the amidine provided Compound 28 (48%,unoptimized), the 4′-chlorobiphenyl derivative of Compound 25. Bydesign, the final reaction introducing the amidine as well as thesequential glycosylation reactions and the reductive amination could beconducted effectively on fully functionalized substrates, lackingprotecting groups and incorporating the vancomycin disaccharide.

Total synthesis of vancomycin, [Ψ[C(═S)NH]Tpg⁴]-vancomycin,[Ψ[C(═NH)NH]Tpg⁴]-vancomycin, and [Ψ[CH₂NH]Tpg⁴]vancomycin and their(4′-chlorobiphenyl)methyl derivatives

The studies piloted with the C-terminus hydroxymethyl derivatives aswell as vancomycin aglycon itself defined the approach taken andprovided the experience needed to address the fully functionalizedresidue 4-modified aglycons. The two sequential glycosylations ofvancomycin aglycon Compound 7 [Nakayama et al., Org. Lett. 2014, 16,3572], the freshly prepared synthetic thioamide Compound 8 [Okano etal., J. Am. Chem. Soc. 2014, 136, 13522], amidine Compound 9 [(a) Xie etal., J. Am. Chem. Soc. 2011, 133, 13946; (b) Xie et al., J. Am. Chem.Soc. 2012, 134, 1284], and the more recently re-prepared methyleneanalogue Compound 10 [Crowley et al., J. Am. Chem. Soc. 2006, 128, 2885]were conducted with the recombinant glycosyl-transferases [(a) Losey etal., Biochemistry 2001, 40, 4745; (b) Oberthur et al., J. Am. Chem. Soc.2005, 127, 10747; (c) Losey et al., Chem. Biol. 2002, 9, 1305; (d) Donget al., J. Am. Chem. Soc. 2002, 124, 9064; and (e) Kruger et al., Chem.Biol. 2005, 12, 131] and the synthetic glycosyl donors (UDP-glucose/GtfEand UDP-vancosamine/GtfD) to provide the intermediate pseudoaglycons andsubsequently, vancomycin (Compound 1, 87%) and the fully functionalizedvancomycin analogues bearing single atom changes in the binding pocket,[Ψ[C(═S)NH]Tpg⁴]-vancomycin (Compound 2, 87%, HPLC conversion >95%) and[Ψ[CH₂NH]Tpg⁴]vancomycin (Compound 16, 76%, HPLC conversion >95%).

A comparison of the relative efficiency of the initial glycosylationreaction with Compounds 3 and 5 conducted on an analytical scalealongside vancomycin aglycon (Compound 2). Unlike the significant impactof the C-terminal hydroxymethyl group, but like the well toleratedN-terminus substitutions [(a) Nakayama et al., Org. Lett. 2014, 16,3572; and (b) Dong et al., J. Am. Chem. Soc. 2002, 124, 9064],modifications to the vancomycin binding pocket itself had a minimalimpact on both the rate and overall efficiency of the initialGtfE-catalyzed reaction. However, and like the observations made withthe C-terminal hydroxymethyl amidine, the amidine aglycon Compound 9failed to undergo successful GtfE-catalyzed glycosylation. Althoughsmall amounts of product could be detected by HPLC, the aglycon itselfunderwent competitive conversion to several byproducts under the basicconditions (pH 9) required of the reaction.

The second glycosylation reaction catalyzed by GtfD using theco-substrate UDP-vancosamine proceeded rapidly (<3 hours) regardless ofthe aglycon substrate, displaying no significant impact of the nature ofresidue 4 (amide, thioamide, or methylene) and the conditions requiredno further optimization. For [Ψ[C(═NH)NH]Tpg⁴]vancomycin (Compound 3),direct conversion of thioamide Compound 2 to the corresponding amidine(10 equiv AgOAc, sat. NH₃-MeOH, 25° C., 7 hours) provided Compound 3.

Subsequent introduction of the chlorobiphenyl group with[Ψ[C(═S)NH]Tpg⁴]vancomycin (Compound 2) and [Ψ[CH₂NH]Tpg⁴]vancomycin(Compound 16) by reductive amination (1.5 equiv4-(4-chloro-phenyl)benzaldehyde, 5 equiv i-Pr₂NEt, DMF, 50-70° C., 2hours; 100 equiv NaCNBH₃, 70° C., 5 hours) provided Compound 5 (57%) andCompound 17 (41%) on the unprotected vancomycin analogues withoutoptimization using conditions piloted with 4′-chlorobiphenyl vancomycin(Compound 4, 61-74%) itself [a) Chen et al., Tetrahedron 2002, 58, 6585;and (b) Kerns et al., J. Am. Chem. Soc. 2000, 122, 12608-12609]. DirectAgOAc-promoted (10 equiv, sat. NH₃-MeOH, 25° C., 7 hours) conversion ofthe thioamide Compound 5 to the amidine provided Compound 6 (45%), the4′-chlorobiphenyl derivative of [Ψ[C(═NH)NH]Tpg⁴]-vancomycin (Compound3).

Significantly, the reductive amination was conducted without competingreaction of either the thioamide or the N-terminus and residue 4secondary amines, the entire 3-4 step sequence could be conductedwithout protecting groups, and the amidine introduction was implementedwithout competitive deglycosylation.

It is further worth noting that the enzymatic glycosylations wereconducted on about 1-3 mg of substrate with 1 mol % enzyme and 4 equiv.of UDP-glucose or UDP-vancosamine, reflecting a piloted laboratoryscale. However, the expression and purification of the enzymes and thechemical synthesis of UDP-vancosamine, along with the commercialavailability of UDP-glucose, were conducted on scales that would easilysupport laboratory preparations on much larger scales (about 100-fold)than exemplified herein and are easily scaled beyond even this level.

Specific Syntheses

In a total volume of 2.6 mL, 2.0 mM UDP-vancosamine (2.8 mg, 5.1 μmol)and 0.5 mM Compound 13 (1.7 mg, 1.3 μmol) were incubated with 75 mMTricine-NaOH (pH 9.0), 2 mM tris-(2-carboxyethyl)phosphine, 0.2 mg/mLbovine serum albumin, 1 mM MgCl₂, glycerol (10% v/v), and 10 μM GtfD for3 hours at 37° C. The reaction mixture was quenched by the addition ofMeOH (23 mL) at 0° C. and was passed through a 0.45 μm polyethersulfonemembrane filter and concentrated by evaporation to a final volume ofabout 3 mL. After the addition of H₂O (1.0 mL), the mixture was purifiedby semi-preparative reverse-phase HPLC. For the HPLC, Vydac®218TP1022-C18, 10 μm, 22×250 mm, 1-40% MeCN/H₂O-0.07% TFA gradient over40 minutes, 10 mL/minute, t_(R)=21.3 minute) was used to afford Compound2 (0.97 mg, 52% yield) as a white amorphous solid: ¹H NMR (CD₃OD, 600MHz, 298° K) δ 8.80 (d, J=6.0 Hz, 1H), 8.49 (s, 1H), 7.73-7.72 (m, 1H),7.68-7.63 (m, 5H), 7.32-7.27 (m, 1H), 7.22 (d, J=1.6 Hz, 1H), 6.78 (d,J=8.4 Hz, 1H), 6.46 (d, J=1.6 Hz, 1H), 6.40 (d, J=1.6 Hz, 1H), 6.17 (s,1H), 5.85 (s, 1H), 5.49-5.41 (m, 3H), 5.34-5.32 (m, 5H), 4.45-4.38 (brm, 1H), 4.29 (s, 1H), 4.25-4.21 (m, 1H), 4.08-4.06 (m, 2H), 3.99 (s,1H), 3.92-3.88 (m, 1H), 3.86-3.83 (m, 2H), 3.80-3.78 (m, 1H), 3.70-3.61(m, 2H), 3.59-3.51 (m, 2H), 3.44-3.42 (m, 1H), 3.22-3.20 (m, 1H),3.01-2.98 (m, 1H), 2.77 (s, 3H), 2.36-2.28 (m, 2H), 2.08-2.04 (m, 2H),1.95-1.93 (m, 1H), 1.90-1.85 (m, 1H), 1.71-1.64 (m, 1H), 1.51 (s, 3H),1.45-1.35 (m, 3H), 1.20 (d, J=6.6 Hz, 3H), 1.19-1.12 (m, 1H), 1.00 (d,J=6.0 Hz, 3H), 0.98 (d, J=6.6 Hz, 3H); ESI-TOF HRMS m/z 1464.4131 (M+H⁺,C₆₆H₇₅Cl₂N₉O₂₃S requires 1464.4152).

HPLC was used to separate the crude reaction mixtures in the conversionof Compound 8 to Compound 13, and Compound 13 to Compound 2. ForCompound 8 to Compound 13, Vydac® 218TP1022-C18, 10 μm, 22×250 mm, 1-40%MeCN/H₂O-0.07% TFA gradient over 40 minutes, 10 mL/minute, t_(R)=23.2minutes was used, indicating that the isolated yield (75%)underestimates the extent of the conversion (86-92% by HPLC). ForCompound 13 to Compound 2, Vydac® 218TP1022-C18, 10 μm, 22×250 mm, 1-40%MeCN/H₂O-0.07% TFA gradient over 40 minutes, 10 mL/minute, t_(R)=21.3minutes was used, indicating that the isolated yield (52%)underestimates the extent of the conversion (95-100% by HPLC).

Additional Synthesis

In a total volume of 1.3 mL, 3.0 mM UDP-vancosamine (2.2 mg, 3.8 μmol)and 0.5 mM Compound 13 (0.84 mg, 0.64 μmol) were incubated with 75 mMTricine-NaOH (pH 9.0), 2 mM tris-(2-carboxyethyl)-phosphine, 0.2 mg/mLbovine serum albumin, 1 mM MgCl₂, glycerol (10% v/v), and 5 μM GtfD for1 hour at 37° C. It is noted that this reaction sequence was conductedonly twice on a preparative scale and consequently is not yet optimized.The reaction mixture was quenched by the addition of MeOH (8 mL) at 0°C. and was passed through a 0.45 μm polyethersulfone membrane filter andconcentrated by evaporation to a final volume of about 2 mL. After theaddition of H₂O (1.0 mL), the mixture was purified by semi-preparativereverse-phase HPLC as discussed above to afford Compound 2 (0.81 mg, 87%yield) as a white amorphous solid.

Second Additional Synthesis

In a total volume of 1.4 mL, 3.0 mM UDP-vancosamine (2.5 mg, 4.6 μmol)and 0.5 mM Compound 13 (1.1 mg, 0.84 μmol) were incubated with 75 mMTricine-NaOH (pH 9.0), 2 mM tris-(2-carboxyethyl)-phosphine, 0.2 mg/mLbovine serum albumin, 1 mM MgCl₂, glycerol (10% v/v) and 10 μM GtfD for3 hours at 37° C. The reaction mixture was quenched by the addition ofMeOH (10 mL) at 0° C., was passed through a 0.45 μm polyethersulfonemembrane filter and concentrated by evaporation to a final volume ofabout 2 mL. After the addition of H₂O (2.0 mL), the mixture was purifiedby semi-preparative reverse-phase HPLC (Vydac® 218TP1022-C18, 10 μm,22×250 mm, 1-40% MeCN/H₂O about 0.07% TFA gradient over 40 minutes, 10mL/minute, t_(R)=20.7 minutes) to afford Compound 2 (1.0 mg, 84%) as awhite amorphous solid: ¹H NMR (CD₃OD, 600 MHz, 298 K) δ 8.29 (s, 1H),7.73-7.62 (m, 3H), 7.57 (d, 1H, J=9.0 Hz), 7.30 (d, 1H, J=9.0 Hz), 7.26(d, 1H, J=8.4 Hz), 7.18 (s, 1H), 6.96 (d, 1H, J=9.0 Hz), 6.79 (d, 1H,J=8.4 Hz), 6.65 (s, 1H), 6.46-6.42 (m, 3H), 6.07 (s, ¹H), 5.77 (s, 1H),5.44 (d, 1H, J=7.8 Hz), 5.40 (d, 1H, J=4.2 Hz), 5.31 (s, 1H), 5.28-5.25(m, 3H), 4.40-4.39 (br m, 1H), 4.31-4.28 (m, 1H), 4.22-4.20 (br m, 2H),4.07-4.01 (m, 2H), 3.97-3.94 (m, 1H), 3.86 (d, 1H, J=12.0 Hz), 3.82-3.79(m, 1H), 3.76-3.73 (m, 1H), 3.68-3.55 (m, 3H), 3.52-3.50 (m, 1H),2.97-2.94 (m, 1H), 2.76 (s, 3H), 2.07-2.04 (m, 1H), 1.92 (d, 1H, J=13.8Hz), 1.88-1.83 (m, 1H), 1.48 (s, 3H), 1.40-1.29 (m, 2H), 1.19 (d, 3H,J=6.6 Hz), 1.02 (d, 3H, J=6.6 Hz), 1.00 (d, 3H, J=6.6 Hz); ESI-TOF HRMSm/z 1450.4375 (M+H⁺, C₆₆H₇₈Cl₂N₉O₂₂S requires 1450.4353).

See also, Okano et al., J. Am. Chem. Soc. 2014, 136, 13522.

A mixture of Compound 2 (0.27 mg, 0.18 μmol) and AgOAc (0.3 mg, 1.8μmol) was treated with saturated NH₃—CH₃OH (0.2 mL) at 0° C. Thereaction mixture was stirred for 7 hours at 25° C. The reaction mixturewas quenched by the addition of 50% CH₃OH in H₂O (0.2 mL), and theresidue was purified by semi-preparative reverse-phase HPLC. For theHPLC, Zorbax® SB-C18, 5 μm, 9.4×150 mm, 1-40% MeCN/H₂O-0.07% TFAgradient over 40 minutes, 3 mL/minute, t_(R)=16.0 minutes was used toafford Compound 3 as a white amorphous solid: ¹H NMR (CD₃OD, 600 MHz,298° K) δ 7.8-7.68 (m, 3H), 7.43 (s, 1H), 7.20-7.11 (m, 2H), 7.04 (s,1H), 6.89 (s, 1H), 6.49-6.45 (m, 2H), 5.59-5.51 (m, 3H), 5.42-5.38 (m,2H), 4.31-4.16 (m, 3H), 3.82-3.76 (m, 2H), 3.67-3.47 (m, 3H), 3.19 (s,1H), 2.95-2.82 (m, 5H), 2.45-2.34 (m, 1H), 2.11-1.99 (m, 3H), 1.90-1.75(br m, 2H), 1.65 (s, 3H), 1.24-1.19 (m, 5H), 0.88 (d, J=6.0 Hz, 3H),0.83 (d, J=6.0 Hz, 3H); ESI-TOF HRMS m/z 724.2307 (M+2H⁺,C₆₆H₇₆Cl₂N₁₀O₂₃ requires 724.2304).

Additional Synthesis

A mixture of Compound 2 (0.38 mg, 0.26 μmol) and AgOAc (0.43 mg, 2.6μmol) was treated with anhydrous saturated NH₃—CH₃OH (0.2 mL) at 25° C.The reaction mixture was stirred for 6 hours at 25° C. before thesolvent was removed under a stream of N₂. The residue was dissolved in50% CH₃OH in H₂O (0.2 mL) and purified by semi-preparative reverse-phaseHPLC (Zorbax® SB-C18, 5 μm, 9.4×150 mm, 1-40% MeCN/H₂O-0.07% TFAgradient over 40 minutes, 3 mL/minute, t_(R)=16.4 minutes) to affordCompound 3 (86 μg, 50% yield brsm, unoptimized) as a white film: ¹H NMR(CD₃OD, 600 MHz, 298 K) δ 7.74 (d, J=9.0 Hz, 1H), 7.74 (s, 1H), 6.90 (d,J=8.4 Hz, 1H), 6.68 (s, 1H), 6.44 (s, 1H), 5.52 (d, J=11.2 Hz, 1H),5.45-5.37 (m, 5H), 4.33 (s, 1H), 4.13-4.06 (m, 2H), 3.85 (s, 1H), 3.77(d, J=9.0 Hz, 1H), 3.67-3.52 (m, 2H), 2.88 (s, 3H), 2.45-2.41 (m, 1H),2.07 (d, J=10.8 Hz, 1H), 1.86 (s, 1H), 1.61 (s, 1H), 1.51-1.39 (m, 3H),1.30 (s, 1H), 1.28-1.20 (m, 3H), 0.89 (d, J=6.0 Hz, 3H), 0.85 (d, J=6.0Hz, 3H); ESI-TOF HRMS m/z 1433.4760 (M+H⁺, C₆₆H₇₈Cl₂N₁₀O₂₂ requires1433.4742).

See also, Okano et al., J. Am. Chem. Soc. 2014, 136, 13522.

A solution of Compound 1 (vancomycin, 0.45 mg, 0.31 μmol) in anhydrousDMF (30 μL) was treated with 4-(4′-chlorophenyl)benzaldehyde (0.1 M inDMF, 4.7 μL, 0.47 μmol) and i-Pr₂NEt (distilled, 0.1 M in DMF, 15.6 μL,1.56 μmol) at 25° C. The reaction mixture was stirred for 2 hours at 70°C. After the reaction was complete, the mixture was treated with NaCNBH₃(1 M in THF, 31.2 μL, 31.2 μmol) and stirred for 5 hours at 70° C. Thereaction mixture was quenched by the addition of 50% CH₃OH in H₂O (0.2mL) at 25° C. and the residue was purified by semi-preparativereverse-phase HPLC. For the HPLC, Zorbax® SB-C18, 5 μm, 9.4×150 mm,1-40% MeCN/H₂O-0.07% TFA gradient over 40 minutes, 3 mL/minute,t_(R)=34.3 minutes) was used to afford Compound 4 (0.31 mg, 61% yield)as a white amorphous solid: ¹H NMR (CD₃OD, 600 MHz, 298° K) δ 8.98 (s,1H), 8.71 (s, 1H), 7.76-7.70 (m, 5H), 7.62 (d, J=8.4 Hz, 2H), 7.56 (d,J=8.4 Hz, 2H), 7.45 (d, J=8.4 Hz, 2H), 7.20 (d, J=9.0 Hz, 1H), 7.08 (s,1H), 6.71 (br s, 1H), 6.52 (d, J=2.4 Hz, 1H), 6.41 (d, J=2.4 Hz, 1H),5.63 (s, 1H), 5.52 (s, 1H), 5.40-5.37 (m, 2H), 5.28 (d, J 2.4 Hz, 1H),4.77 (s, 1H), 4.73 (d, J=6.0 Hz, 1H), 4.27 (s, 1H), 4.19-4.15 (m, 3H),4.08-3.95 (m, 2H), 3.90-3.80 (m, 2H), 3.68-3.62 (m, 3H), 3.43 (s, 1H),2.92 (d, J=12.6 Hz, 1H), 2.78 (s, 1H), 2.19 (d, J=12.0 Hz, 1H), 2.05 (d,J=13.2 Hz, 1H), 1.90-1.87 (m, 1H), 1.88 (s, 3H), 1.68-1.65 (m, 1H), 1.25(d, J=6.6 Hz, 3H), 0.95-0.92 (m, 6H); ESI-TOF HRMS m/z 824.7421 (M+2H⁺,C₇₉H₈₄Cl₃N₉O₂₄ requires 824.7420).

This reaction was run on scales of 0.2-1.2 mg (55-61% yield) as part ofthe optimization of conditions for use with Compound 5 on the amountsavailable.

Larger Scale Procedure:

A solution of Compound 1 (vancomycin, 90.0 mg, 62.1 μmol) in anhydrousDMF (8.0 mL) was treated with 4-(4′-chlorophenyl)benzaldehyde (19.8 mg,74.5 μmol) and i-Pr₂NEt (51.0 μL, 0.32 mmol) at 25° C. The reactionmixture was stirred for 2 hours at 70° C. After the reaction wascomplete, the mixture was treated with NaCNBH₃ (1 M in THF, 0.32 mL,0.32 mmol) and stirred for 5 hours at 70° C. The reaction mixture wasquenched by the addition of 50% CH₃OH in H₂O (1.0 mL) at 25° C., and theresidue was purified by semi-preparative reverse-phase HPLC. For HPLC,Zorbax® SB-C18, 5 μm, 9.4×150 mm, 1-40% MeCN/H₂O-0.07% TFA gradient over40 minutes, 3 mL/minute, t_(R)=34.3 minutes was used to afford Compound4 (75.3 mg, 74% yield) as a white amorphous solid.

Additional Synthesis

A solution of Compound 1 (1.0 mg, 0.65 μmol) in anhydrous DMF (0.1 mL)was treated with 4-(4-chlorophenyl)benzaldehyde (0.1 M in DMF, 9.7 μL,0.97 μmol) and i-Pr₂NEt (distilled, 0.1 M in DMF, 32.3 μL, 3.23 μmol) at25° C. The reaction mixture was stirred for 12 hours at 30° C. After thereaction was complete, the mixture was treated with NaBH(OAc)₃ (13.7 mg,64.6 μmol) and stirred for 2 hours at 30° C.

The reaction mixture was quenched by the addition of 50% CH₃OH in H₂O(0.2 mL) at 25° C. and the residue was purified by semi-preparativereverse-phase HPLC (Zorbax® SB-C18, 5 μm, 9.4×150 mm, 1-40%MeCN/H₂O-0.07% TFA gradient over 40 minutes, 3 mL/minute, t_(R)=35.2minutes) to afford Compound 4 (0.73 mg, 67% yield) as a white amorphoussolid: ¹H NMR (CD₃OD, 600 MHz, 298K) δ 7.71-7.68 (m, 3H), 7.66-7.60 (m,5H), 7.57-7.54 (m, 2H), 7.47-7.43 (m, 3H), 7.32 (d, 1H, J=8.4 Hz), 7.29(d, 1H, J=7.8 Hz), 7.11 (d, 1H, J=8.4 Hz), 6.94 (d, 1H, J=8.4 Hz),6.45-6.43 (m, 1H), 6.41-6.37 (m, 1H), 6.35 (d, 1H, J=2.4 Hz), 6.33 (d,1H, J=2.4 Hz), 6.41-6.37 (m, 1H), 6.34-6.33 (m, 1H), 5.74 (d, 1H, J=11.2Hz), 5.60 (d, 1H, J=18.0 Hz), 5.56 (d, 1H, J=7.8 Hz), 5.53 (d, 1H,J=12.0 Hz), 5.49 (d, 1H, J=4.2 Hz), 5.44-5.42 (m, 1H), 4.55-4.53 (m,1H), 4.22-4.15 (m, 1H), 4.13-4.05 (m, 1H), 3.95-3.93 (m, 1H), 3.86-3.84(m, 2H), 3.75-3.72 (m, 1H), 3.65 (br s, 1H), 3.62 (d, 1H, J=9.6 Hz),3.53-3.50 (m, 1H), 2.78 (s, 3H), 2.52-2.48 (m, 1H), 2.21-2.16 (m, 1H),2.04 (d, 1H, J=13.2 Hz), 1.67-1.64 (br m, 5H), 1.32 (d, 3H, J=6.6 Hz),1.03-1.00 (m, 3H), 0.98-0.95 (m, 3H); ESI-TOF HRMS m/z 1634.5057 (M+H⁺,C₇₉H₈₇Cl₃N₉O₂₃ requires 1634.4975).

See also, Okano et al., J. Am. Chem. Soc. 2014, 136, 13522.

Following the procedure detailed for Compound 4 and using Compound 2 asthe starting material, (0.42 mg, 0.29 μmol), semi-preparativereverse-phase HPLC was used to purify the compound. For the HPLC,Zorbax® SB-C18, 5 μm, 9.4×150 mm, 1-40% MeCN/H₂O-0.07% TFA gradient over40 minutes, 3 mL/minute, t_(R)=34.9 minutes afforded Compound 5 (0.27mg, 57% yield) as a white amorphous solid: ¹H NMR (CD₃OD, 600 MHz, 298°K) δ 7.72-7.66 (m, 6H), 7.62 (d, J=8.4 Hz, 2H), 7.55 (d, J=8.4 Hz, 2H),7.34 (s, 1H), 7.27 (d, J=8.4 Hz, 2H), 7.17 (s, 1H), 6.75 (d, J=9.0 Hz,1H), 6.52 (d, J=2.4 Hz, 1H), 6.39 (d, J=2.4 Hz, 1H), 5.52-5.46 (m, 2H),5.37-5.31 (m, 3H), 4.59 (s, 1H), 4.24 (s, 1H), 4.16 (d, J=12.6 Hz, 1H),4.07 (d, J=12.6 Hz, 1H), 3.92 (d, J=6.0 Hz, 1H), 3.85-3.75 (m, 2H), 3.63(dd, J=9.0, 9.0 Hz, 1H), 3.60-3.56 (m, 2H), 3.44-3.39 (m, 2H), 3.19 (s,1H), 2.72 (s, 3H), 2.40-2.25 (m, 1H), 2.20-2.15 (m, 1H), 2.10-1.97 (m,2H), 1.83-1.73 (m, 2H), 1.69-1.59 (m, 5H), 1.25 (d, J=6.6 Hz, 3H),1.00-0.94 (m, 6H); ESI-TOF HRMS m/z 832.7286 (M+2H⁺, C₇₉H₈₄Cl₃N₉O₂₃Srequires 832.7306).

Additional Synthesis

A solution of Compound 2 (0.62 mg, 0.42 μmol) in anhydrous DMF (30 μL)was treated with 4-(4-chlorophenyl)benzaldehyde (0.1 mM in DMF, 5.5 μL,0.546 μmol) and i-Pr₂NEt (distilled, 0.1 mM in DMF, 21 μL, 2.1 μmol) at25° C. The reaction mixture was stirred for 9 hours at 30° C. After thereaction was complete, the mixture was treated with NaBH(OAc)₃ (11.2 mg,42.0 μmol) and stirred for 2 hours at 30° C. The reaction mixture wasquenched with the addition of 50% CH₃OH in H₂O (0.2 mL) and the residuewas purified by semi-preparative reverse-phase HPLC (1-40%MeCN/H₂O-0.07% TFA isocratic gradient over 40 minutes) to affordCompound 5 (0.52 mg, 74%) as a white amorphous solid: ¹H NMR (CD₃OD, 600MHz) δ 8.30 (d, 1H, J=6.6 Hz), 7.72-7.67 (m, 5H), 7.63 (d, 2H, J=8.4Hz), 7.60 (d, 1H, J=6.0 Hz), 7.56 (d, 2H, J=7.8 Hz), 7.47 (d, 2H, J=8.4Hz), 7.33 (d, 1H, J 8.4 Hz), 7.29 (d, 1H, J=8.4 Hz), 7.19 (d, 1H, J=2.4Hz), 6.99-6.97 (m, 1H), 6.80 (d, 1H, J=8.4 Hz), 6.56 (d, 1H, J=2.4 Hz),6.42 (d, 1H, J=1.8 Hz), 6.13-6.08 (br m, 1H), 5.80 (s, 1H), 5.51 (d, 1H,J 7.2 Hz), 5.46 (d, 1H, J=4.8 Hz), 5.33 (d, 1H, J=3.0 Hz), 5.31-5.29 (brm, 2H), 5.27 (s, 1H), 4.40-4.39 (m, 1H), 4.33-4.30 (m, 1H), 4.24 (s,1H), 4.16 (d, 1H, J=12.0 Hz), 4.08-4.02 (m, 3H), 3.98-3.95 (m, 1H),3.90-3.82 (m, 2H), 3.77-3.75 (m, 1H), 3.64-3.60 (m, 2H), 3.54-3.50 (m,2H), 3.44-3.42 (m, 1H), 3.20-3.19 (m, 1H), 2.97 (d, 1H, J=13.8 Hz), 2.77(s, 3H), 2.36-2.32 (m, 1H), 2.20-2.16 (m, 1H), 2.02 (d, 1H, J=13.8 Hz),1.89-1.84 (m, 1H), 1.81-1.76 (m, 1H), 1.71-1.68 (m, 1H), 1.66 (s, 3H),1.27 (d, 3H, J=6.6 Hz), 1.03 (d, 3H, J=6.0 Hz), 1.00 (d, 3H, J=6.6 Hz);ESI-TOF HRMS m/z 825.7447 (M+2H⁺, C₇₉H₈₆Cl₃N₉O₂₃ requires 825.7410).

See also, Okano et al., J. Am. Chem. Soc. 2014, 136, 13522.

Following the procedure detailed for Compound 3 and using Compound 5 asthe starting material, (0.31 mg, 0.19 μmol), semi-preparativereverse-phase HPLC was used for purification. For the HPLC, Zorbax®SB-C18, 5 μm, 9.4×150 mm, 1-40% MeCN/H₂O-0.07% TFA gradient over 40minutes, 3 mL/minute, t_(R)=33.6 minutes afforded Compound 6 as a whiteamorphous solid: ¹H NMR (CD₃OD, 600 MHz, 298° K) δ 7.82-7.72 (m, 3H),7.64 (d, J=8.4 Hz, 2H), 7.56 (d, J=8.4 Hz, 2H), 7.49-7.33 (m, 4H), 7.07(s, 1H), 6.91 (d, J=9.0 Hz, 1H), 6.51-6.46 (m, 2H), 5.61-5.37 (m, 5H),4.33 (br s, 1H), 4.21-4.13 (m, 2H), 4.09-4.03 (m, 2H), 3.88-3.75 (m,2H), 3.73-3.58 (m, 5H), 3.51-3.49 (m, 1H), 3.37 (s, 1H), 3.21 (s, 1H),2.88 (s, 3H), 2.81-2.76 (m, 2H), 2.49-2.45 (m, 1H), 2.42-2.28 (m, 2H),2.21-2.06 (m, 3H), 1.85-1.77 (m, 2H), 1.65 (s, 3H), 1.40-1.30 (m, 4H),0.92 (d, J=6.6 Hz, 3H), 0.88 (d, J=6.6 Hz, 3H); ESI-TOF HRMS m/z824.2539 (M+2H⁺, C₇₉H₈₈Cl₃N₁₀O₂₃ requires 824.2578)

Additional Synthesis

A mixture of Compound 5 (0.35 mg, 0.20 μmol) and AgOAc (0.33 mg, 2.0μmol) was treated with anhydrous saturated NH₃—CH₃OH (0.2 mL) at 25° C.The reaction mixture was stirred for 6 hours at 25° C. before thesolvent was removed under a stream of N₂. The residue was dissolved in50% CH₃OH in H₂O (0.2 mL) and purified by semi-preparative reverse-phaseHPLC (Zorbax® SB-C18, 5 μm, 9.4×150 mm, 1-40% MeCN/H₂O-0.07% TFAgradient over 40 minutes, 3 mL/minute, t_(R)=33.2 minutes) to affordCompound 6 (86 μg, 48% yield brsm, unoptimized) as a white film: ¹H NMR(CD₃OD, 600 MHz, 298 K) δ 7.79-7.68 (m, 3H), 7.61 (d, J=8.4 Hz, 2H),7.54 (d, J=7.8 Hz, 2H), 7.46-7.44 (m, 4H), 7.08-7.02 (br m, 2H), 6.88(d, J=9.0 Hz, 1H), 6.66 (s, 1H), 6.43 (d, J=2.4 Hz, 1H), 5.58-5.48 (m,2H), 5.44-5.40 (m, 3H), 4.37-4.28 (m, 1H), 4.18-4.15 (m, 2H), 4.11 (d,J=4.2 Hz, 1H), 4.09-4.02 (m, 4H), 3.88-3.75 (m, 2H), 3.67-3.54 (m, 4H),2.87 (s, 3H), 2.74 (br s, 1H), 2.41 (dd, J=14.4, 4.8 Hz, 1H), 2.19-2.16(m, 1H), 2.06 (s, 1H), 2.03 (s, 1H), 1.85 (br s, 1H), 1.65-1.55 (m, 4H),1.30-1.29 (m, 4H), 1.22-1.19 (m, 1H), 0.88 (d, J=6.0 Hz, 3H), 0.84 (d,J=6.6 Hz, 3H); ESI-TOF HRMS m/z 817.2614 (M+2H⁺, C₇₉H₈₈Cl₃N₁₀O₂₂requires 817.2604).

See also, Okano et al., J. Am. Chem. Soc. 2014, 136, 13522.

Conversion of Compound A to Compound B

The reaction was performed on scales ranging from 3.2 to about 8.0 mg(50 to about 59%, 3 steps). A representative procedure follows: Asolution of Compound A [Crowley et al., J. Am. Chem. Soc. 2006, 128,2885] (5.9 mg, 4.3 μmol) in acetone (0.15 mL, degassed) and saturatedaqueous NH₄Cl (20 μL, degassed) was treated with zinc nanoparticle(Aldrich, 11.0 mg, 0.17 mmol) at 25° C. The reaction mixture was stirredat 25° C. for 2 hours before the solvent was removed under a stream ofN₂.

The residue was dissolved in EtOAc and purified through a short plug ofsilica gel (100% EtOAc then 10% CH₃OH—CH₂Cl₂) to afford thecorresponding aniline as a white amorphous crude solid. This solid wasdissolved in MeCN (degassed, 0.3 mL) and treated with HBF₄ (0.1 M inMeCN, 43 μL, 4.3 μmol) at 0° C. The reaction mixture was stirred for 3minutes before the drop-wise addition of t-butylnitrite (0.1 M in MeCN,43 μL, 4.3 μmol) at 0° C. The reaction mixture was stirred at 0° C. for3 minutes before an aqueous mixture (degassed, 0.4 mL) containing CuCl(9.0 mg, 86 μmol) and CuCl₂ (15.3 mg, 108 μmol) was transferred to theabove solution in one portion at 0° C. The heterogeneous mixture waspermitted to warm to 25° C. and stirred for 45 minutes.

The reaction mixture was purified by PTLC (SiO₂, 10% CH₃OH—CH₂Cl₂)afforded the corresponding aryl chloride as a white amorphous solid.This solid was dissolved in anhydrous MeCN (degassed, 0.3 mL) andtreated with N-methyl-N-tert-butyldimethylsilyl-trifluoroacetamide(MTBSTFA; Sigma-Aldrich, 43 μL, 1.8 mmol). The reaction mixture waswarmed to 55° C. and stirred for 24 hours. This protocol was repeatedfor a second 5.9 mg of Compound A and the batches were later combinedfor work-up.

The reaction mixture was cooled to 25° C. and the solvent was removedunder a stream of N₂. The residue was diluted with EtOAc (0.5 mL), 0.1 NHCl (0.5 mL) was added, and the mixture was stirred for 30 minutes. Thelayers were separated, and the aqueous layer was extracted with EtOAc(3×0.5 mL). The combined organic layers were washed with saturatedaqueous NaCl (0.5 mL), dried (Na₂SO₄) and the solvent was removed underreduced pressure. PTLC (SiO₂, 4% CH₃OH—CH₂Cl₂) afforded Compound B (6.7mg, 51%, 3 steps) as a white amorphous solid identical in all respectswith authentic material (¹H NMR, CD₂OD) [Crowley et al., J. Am. Chem.Soc. 2006, 128, 2885].

Improved Protocol for the Sandmeyer Chemistry Used in the Conversion ofCompound C to Compound D

The reaction was performed on scales ranging from 3.2-4.0 mg (55-60%, 3steps). A representative procedure follows: A solution of Compound C[(a) Xie et al., J. Am. Chem. Soc. 2011, 133, 13946; and (b) Xie et al.,J. Am. Chem. Soc. 2012, 134, 1284] (3.2 mg, 2.3 μmol) in acetone (0.15mL, degassed) and saturated aqueous NH₄Cl (20 μL, degassed) was treatedwith zinc nanoparticle (Sigma-Aldrich, 8.9 mg, 0.14 mmol) at 25° C. Thereaction mixture was stirred at 25° C. for 0.5 hours before the solventwas removed under a stream of N₂. The residue was dissolved in EtOAc andpurified through a short plug of silica gel (100% EtOAc then 12%CH₃OH—CH₂Cl₂) to afford the corresponding aniline as a white amorphouscrude solid. This solid was dissolved in MeCN (degassed, 250 μL) andtreated with HBF₄ (0.1 M in MeCN, 26 μL, 2.6 μmol) at −15° C. Thereaction mixture was stirred for 3 minutes before the drop-wise additionof t-butylnitrite (0.1 M in MeCN, 26 μL, 2.6 μmol) at −15° C.

The reaction mixture was stirred at −15° C. for 20 minutes before anaqueous mixture (degassed, 0.3 mL) containing CuCl (11.2 mg, 114 μmol)and CuCl₂ (21.0 mg, 157 μmol) was transferred to the above solution inone portion at −30° C. The heterogeneous mixture was permitted to warmto 25° C. and stirred for 0.5 hours. The reaction mixture was directlypurified by PTLC (SiO₂, 10% CH₃OH—CH₂Cl₂) and afforded the nitro groupconverted to a chloro group-containing Compound C-1 as a white amorphoussolid.

Compound C-1 was dissolved in anhydrous MeCN (degassed, 0.3 mL) andtreated with MTBSTFA (53 μL, 0.22 mmol). The reaction mixture was warmedto 55° C. and stirred for 24 hours. The reaction mixture was cooled to25° C. and the solvent was removed under a stream of N₂. The residue wasdiluted with EtOAc (0.5 mL), 0.1 N HCl (0.5 mL) was added, and themixture was stirred for 30 minutes.

The layers were separated, and the aqueous layer was extracted withEtOAc (3×0.5 mL). The combined organic layers were washed with saturatedaqueous NaCl (0.5 mL), dried (Na₂SO₄) and the solvent was removed underreduced pressure. PTLC (SiO₂, 4% CH₃OH—CH₂Cl₂) afforded Compound D (2.2mg, 60%, 3 steps) as a white amorphous solid identical in all respectswith authentic material (¹H NMR, acetone-d₆) [Okano et al., J. Am. Chem.Soc. 2012, 134, 8790].

Improved Protocol for Jones Oxidation and Global Deprotection ConvertingCompound E to Compound 8

This reaction was performed on scales ranging from 0.9-1.8 mg (44-52%, 3steps). A representative procedure follows: A solution of CrO₃(Sigma-Aldrich 99.99%, 17.9 mg) in H₂O (degassed, 340 μL) was treatedwith conc. H₂SO₄ (Sigma-Aldrich 99.999%, 30 μL) at 25° C. An aliquot ofthis stock solution (4.1 μL, 2.2 μmol) was added into a solution ofCompound E [Crowley et al., J. Am. Chem. Soc. 2006, 128, 2885] (1.1 mg,0.73 μmol) in acetone (Sigma-Aldrich HPLC grade, degassed, 78 μL) at 25°C. The reaction mixture was stirred at 25° C. for 24 hours, cooled to 0°C., and quenched by the addition of saturated NH₃—CH₃OH (0.2 mL) anddiluted with anhydrous CH₂Cl₂ (0.2 mL).

The residue was purified through a short plug of silica gel (15%CH₃OH—CH₂Cl₂) to afford the corresponding carboxylic acid as a whiteamorphous crude solid. This solid was treated with TFA (neat, 0.2 mL) at25° C. and stirred at 25° C. for 12 hours.

TFA was removed under a stream of N₂ and the residue was dissolved inMeOH (HPLC grade, 0.3 mL) at 25° C. The reaction mixture was stirred at25° C. for 3 hours before the MeOH was removed under a stream of N₂. Theresidue was treated with AlBr₃ (Aldrich, 192 mg, 0.73 mmol) and EtSH (10μL) at 25° C. and stirred at 25° C. for 72 hours.

The reaction mixture was quenched by the addition of 50% MeOH in H₂O (1mL) at 0° C. and purified by short reverse phase silica gelchromatography (C18-SiO₂, 50% CH₃CN—H₂O) and subsequent semi-preparativereverse-phase HPLC (Zorbax® SB-C18, 5 μm, 9.4×150 mm, 1-40%MeCN/H₂O-0.07% TFA gradient over 40 minutes, 3 mL/minute, t_(R)=27.3minutes) to afford Compound 8 (0.41 mg, 48% yield, 3 steps) as a whiteamorphous solid identical in all respects with authentic material (¹HNMR, CD₃OD) [Crowley et al., J. Am. Chem. Soc. 2006, 128, 2885].

A solution of Compound F [(a) Xie et al., J. Am. Chem. Soc. 2011, 133,13946; and (b) Xie et al., J. Am. Chem. Soc. 2012, 134, 1284] (2.3 mg,1.6 μmol) in DMSO (160 μL) was treated sequentially with H₂O₂ (50%aqueous solution, 12 μL, 98.4 μmol) and K₂CO₃ (10% aqueous solution, 20μL, 16.1 μmol) at 25° C. and the resulting mixture was stirred for 2hours at 25° C. After this time, the reaction mixture was quenched bythe addition of 0.1 N HCl (0.5 mL), and the aqueous phase extracted withEtOAc (3×0.5 mL). The combined organic layers were washed with saturatedaqueous NaCl (0.5 mL), dried (Na₂SO₄) and the solvent was removed underreduced pressure.

PTLC (SiO₂, 12% CH₃OH—CH₂Cl₂) afforded Compound G (1.9 mg, 81%) as awhite amorphous solid: ¹H NMR (CD₃OD, 600 MHz, 298 K) mixture of tworotamers (rotamer A:B=4:1) δ (for rotamer A) 8.42 (d, 1H, J=6.6 Hz),7.81 (d, 1H, J=7.8 Hz), 7.65 (s, 1H), 7.59 (d, 1H, J=6.0 Hz), 7.45 (d,1H, J=8.4 Hz), 7.34 (d, 1H, J=8.4 Hz), 7.31-7.29 (m, 3H), 7.00 (d, 1H,J=2.4 Hz), 6.97 (s, 1H), 6.94-6.92 (br m, 2H), 6.65 (d, 1H, J=2.4 Hz),5.77 (s, 1H), 5.57-5.52 (m, 1H), 5.51 (s, 1H), 5.37 (d, 1H, J=5.4 Hz),5.28 (s, 1H), 4.97 (d, 1H, J=9.0 Hz), 4.72 (s, 2H), 4.38-4.35 (m, 1H),4.14 (s, 3H), 4.02 (dd, 1H, J=7.8, 7.8 Hz), 3.91 (s, 3H), 3.77-3.71 (m,5H), 3.69 (s, 3H), 3.57 (s, 3H), 3.47 (dd, 1H, J=4.2, 4.2 Hz), 3.39 (s,3H), 3.13 (s, 1H), 2.82 (s, 3H), 2.66-2.62 (m, 1H), 2.53-2.48 (m, 1H),1.90-1.81 (m, 1H), 1.58 (s, 9H), 1.31 (s, 1H), 0.98 (d, 3H, J=6.0 Hz),0.92 (d, 3H, J=6.0 Hz); ESI-TOF HRMS m/z 1417.5045 (M+H⁺, C₆₈H₈₂Cl₂N₈O₂₁requires 1417.5044).

A vial charged with Compound G (1.9 mg, 1.4 μmol) was treated with AlBr₃(35.7 mg, 0.14 mmol) in EtSH (15 μL) at 25° C. and the resulting mixturewas stirred for 8 hours at 25° C. After this time, the reaction mixturewas quenched with the addition of 50% CH₃OH in H₂O (0.5 mL) at 0° C. andthe solvent was removed under a stream of N₂. The residue was dissolvedin 50% CH₃OH in H₂O (0.3 mL) and purified by semi-preparativereverse-phase HPLC (Zorbax® SB-C18, 5 μm, 9.4×150 mm, 1-20%MeCN/H₂O-0.07% TFA gradient over 10 minutes, 3 mL/minute, t_(R)=16.2minute) to afford Compound 10 (1.0 mg, 65%) as a white amorphous solid:¹H NMR (CD₃OD, 600 MHz, 298 K) δ 8.07 (d, 1H, J=8.4 Hz), 7.84 (d, 1H,J=8.4 Hz), 7.75 (d, 1H, J=8.4 Hz), 7.53 (s, 1H), 7.42 (d, 1H, J=8.4 Hz),7.30 (s, 1H), 7.28 (d, 1H, J=8.4 Hz), 7.13 (d, 1H, J=6.0 Hz), 7.07 (s,1H), 6.92 (d, 1H, J=8.4 Hz), 6.67 (s, 1H), 6.44 (s, 1H), 5.42 (s, 1H),5.33 (s, 1H), 4.51-4.43 (m, 3H), 4.35-4.29 (br m, 1H), 4.10 (s, 1H),4.04-4.01 (m, 1H), 3.89-3.85 (m, 1H), 2.77 (s, 3H), 2.66 (d, 1H, J=4.2Hz), 2.03-2.02 (m, 1H), 1.84-1.77 (m, 1H), 1.65-1.62 (m, 1H), 1.31 (s,1H), 1.00 (d, 3H, J=6.0 Hz), 0.94 (d, 3H, J=6.0 Hz); ESI-TOF HRMS m/z1115.3313 (M+H⁺, C₅₃H₅₆Cl₂N₈O₁₅ requires 1115.3315).

See, Nakayama et al., Org. Lett. 2014, 16, 3572.

In a total volume of 1.0 mL, 4.0 mM UDP-glucose (2.3 mg, Sigma-Aldrich,4.0 μmol) and 0.5 mM Compound 8 (0.58 mg, 0.50 μmol) were incubated with75 mM Tricine-NaOH (pH 9.0), 2 mM tris-(2-carboxyethyl)phosphine, 1 mMMgCl₂, glycerol (10% v/v) and 10 μM GtfE for 42 hours at 37° C. Thereaction mixture was quenched by the addition of MeOH (9.0 mL) at 0° C.and the residue was passed through a 0.45 μm polyethersulfone membranefilter and concentrated by evaporation to a final volume of about 1.5mL. After the addition of H₂O (0.5 mL), the mixture was purified bysemi-preparative reverse-phase HPLC. For HPLC, Vydac® 218TP1022-C18, 10μm, 22×250 mm, 1-40% MeCN/H₂O-0.07% TFA gradient over 40 minutes, 10mL/minute, t_(R)=23.2 minutes was used to afford Compound 13 (0.48 mg,75% yield) as a white amorphous solid: ¹H NMR (CD₃OD, 600 MHz, 298° K) δ8.83 (d, J=6.0 Hz, 1H), 8.42 (br s, 1H), 7.74-7.72 (m, 2H), 7.69-7.65(m, 2H), 7.64-7.60 (m, 2H), 7.30 (d, J=8.4 Hz, 1H), 7.24 (br s, 1H),6.78-6.73 (m, 1H), 6.45 (d, J=1.6 Hz, 1H), 6.40 (d, J=1.6 Hz, 1H), 6.26(s, 1H), 5.95 (s, 1H), 5.41-5.36 (m, 3H), 5.32-5.28 (m, 1H), 4.41 (d,J=9.0 Hz, 1H), 4.30 (s, 1H), 4.23 (dd, J=4.8, 4.8 Hz, 1H), 4.07-4.04 (m,1H), 3.92 (d, J=11.4 Hz, 1H), 3.82-3.81 (br m, 1H), 3.68-3.64 (m, 1H),3.57-3.49 (m, 2H), 3.44-3.42 (m, 2H), 3.21-3.20 (m, 1H), 2.79 (s, 3H),2.78 (s, 1H), 2.67 (s, 1H), 1.90-1.84 (m, 2H), 1.74-1.62 (m, 1H),1.46-1.43 (m, 1H), 1.40-1.30 (m, 3H), 0.97 (d, J=6.0 Hz, 3H), 0.95 (d,J=6.0 Hz, 3H); ESI-TOF HRMS m/z 1321.3245 (M+H⁺, C₅₉H₆₂Cl₂N₈O₂₁Srequires 1321.3206).

Additional Synthesis

In a total volume of 10.3 mL, 2.0 mM UDP-glucose (21.7 mg,Sigma-Aldrich, 38.3 μmol) and 0.5 mM Compound 8 (5.5 mg, 4.8 μmol) wereincubated with 75 mM Tricine-NaOH (pH 9.0), 2 mMtris(2-carboxy-ethyl)phosphine, 1 mM MgCl₂, glycerol (25% v/v) and 25 μMGtfE for 38 hours at 37° C. The reaction mixture was quenched by theaddition of MeOH (90 mL) and the residue was passed through a 0.45 μmpolyethersulfone membrane filter and concentrated by evaporation to afinal volume of about 2 mL. After the addition of H₂O (1.0 mL), themixture was purified by semi-preparative reverse-phase HPLC (Vydac®218TP1022-C18, 10 μm, 22×250 mm, 1-40% MeCN/H₂O-0.07% TFA gradient over40 minutes, 10 mL/minute, t_(R)=22.7 minutes) to afford Compound 13 (2.3mg, 35%) as a white amorphous solid and recovered starting materialCompound 8 (2.5 mg, 46%) as a white amorphous solid: ¹H NMR (CD₃OD, 600MHz, 298 K) δ 8.35 (d, 1H, J=6.6 Hz), 7.67-7.66 (m, 2H), 7.64-7.60 (m,2H), 7.35-7.30 (m, 2H), 7.18 (d, 1H, J=2.4 Hz), 6.74 (d, 1H, J=9.0 Hz),6.65 (d, 1H, J=2.4 Hz), 6.44-6.43 (m, 1H), 6.41 (d, 1H, J=2.4 Hz), 6.16(s, 1H), 5.86 (s, 1H), 5.38 (d, 1H, J=7.8 Hz), 5.32-5.29 (m, 3H), 5.26(br s, 1H), 4.40 (d, 1H, J=6.6 Hz), 4.30-4.27 (m, 1H), 4.23-4.21 (m,2H), 4.04-4.01 (m, 2H), 3.97-3.95 (m, 1H), 3.88-3.86 (m, 1H), 3.77-3.74(m, 1H), 3.64-3.62 (m, 1H), 3.50-3.48 (m, 2H), 3.42-3.38 (m, 3H), 3.34(s, 1H), 3.19-3.18 (m, 2H), 3.05-3.00 (m, 1H), 2.75 (s, 3H), 2.30-2.22(m, 1H), 1.90-1.85 (m, 1H), 1.76-1.65 (m, 3H), 1.46-1.40 (m, 1H),1.38-1.36 (m, 1H), 1.00 (d, 3H, J=6.0 Hz), 0.97 (d, 3H, J=6.0 Hz);ESI-TOF HRMS m/z 1307.3420 (M+H⁺, C₅₉H₆₅Cl₂N₈O₂₀S requires 1307.3407).

See also, Okano et al., J. Am. Chem. Soc. 2014, 136, 13522.

In a total volume of 2.8 mL, 2.0 mM UDP-glucose (3.22 mg, Sigma-Aldrich,5.7 μmol) and 0.5 mM of Compound 10 [Okano et al., J. Am. Chem. Soc.2012, 134, 8790] (1.5 mg, 1.3 μmol) were incubated with 75 mMTricine-NaOH (pH 9.0), 2 mM tris-(2-carboxy-ethyl)phosphine, 1 mM MgCl₂,glycerol (25% v/v) and 25 μM GtfE for 48 hours at 37° C. The reactionmixture was quenched by the addition of MeOH (26 mL) and the residue waspassed through a 0.45 μm polyethersulfone membrane filter andconcentrated by evaporation to a final volume of about 1.5 mL. After theaddition of H₂O (1.0 mL), the mixture was purified by semi-preparativereverse-phase HPLC (Zorbax® SB-C18, 5 μm, 9.4×150 mm, 1-40%MeCN/H₂O-0.07% TFA gradient over 40 minutes, 3 mL/minutes, t_(R)=16.8minutes) to afford Compound 15 (1.1 mg, 66%; typically 66-72%) as awhite amorphous solid: ¹H NMR (CD₃OD, 600 MHz, 298 K) δ 8.69 (d, 1H,J=8.4 Hz), 8.53 (d, 1H, J=7.2 Hz), 7.82 (dd, 1H, J=8.4, 2.4 Hz), 7.75(dd, 1H, J=8.4, 1.8 Hz), 7.60 (d, 1H, J=1.8 Hz), 7.41 (d, 1H, J=8.4 Hz),7.32 (d, 1H, J=2.4 Hz), 7.28 (d, 1H, J=8.4 Hz), 7.17-7.13 (m, 3H), 6.95(dd, 1H, J=8.4, 2.4 Hz), 6.48 (d, 1H, J=4.8 Hz), 6.43 (d, 1H, J=2.4 Hz),5.45-5.44 (m, 2H), 5.40 (d, 1H, J=7.8 Hz), 5.38 (d, 1H, J=5.4 Hz), 5.16(d, 1H, J=7.8 Hz), 5.11 (d, 1H, J=9.6 Hz), 5.06 (s, 1H), 4.99-4.98 (m,2H), 4.59 (dd, 1H, J=5.4, 5.4 Hz), 4.36 (dd, 1H, J=8.4, 6.0 Hz),4.27-4.25 (m, 2H), 3.89 (dd, 1H, J=4.8, 2.4 Hz), 3.80-3.76 (m, 1H),3.69-3.65 (m, 1H), 3.57-3.52 (m, 3H), 3.47-3.41 (m, 1H), 2.81 (s, 3H),2.76-2.68 (m, 1H), 2.64 (dd, 1H, J=4.8, 2.4 Hz), 2.32-2.30 (m, 1H), 1.82(dd, 1H, J=8.4, 8.4 Hz), 1.66-1.64 (m, 3H), 0.97 (d, 3H, J=6.0 Hz), 0.92(d, 3H, J=6.0 Hz); ESI-TOF HRMS m/z 1291.3625 (M+H⁺, C₅₉H₆₄Cl₂N₈O₂₁requires 1291.3636).

In a total volume of 1.9 mL, 3.0 mM UDP-vancosamine (3.1 mg, 5.7 μmol)and 0.5 mM Compound 15 (1.1 mg, 0.85 μmol) were incubated with 75 mMTricine-NaOH (pH 9.0), 2 mM tris-(2-carboxyethyl)-phosphine, 0.2 mg/mLbovine serum albumin, 1 mM MgCl₂, glycerol (10% v/v) and 10 μM GtfD for1 hour at 37° C. The reaction mixture was quenched by the addition ofMeOH (10 mL) at 0° C. and was passed through a 0.45 μm polyethersulfonemembrane filter and concentrated by evaporation to a final volume ofabout 2 mL.

After the addition of H₂O (2.0 mL), the mixture was purified byreverse-phase HPLC (Zorbax® SB-C18, 5 μm, 9.4×150 mm, 1-40%MeCN/H₂O-0.07% TFA gradient over 40 minutes, 3 mL/minute, t_(R)=14.0minutes) to afford Compound 16 (0.93 mg, 76%) as a white amorphoussolid: ¹H NMR (CD₃OD, 600 MHz, 298 K) δ 8.65 (d, 1H, J=7.8 Hz), 8.49 (d,1H, J=7.8 Hz), 7.84 (d, 1H, J=9.6 Hz), 7.77 (d, 1H, J=8.4 Hz), 7.62 (d,1H, J=1.6 Hz), 7.41 (d, 1H, J=8.4 Hz), 7.33 (d, 1H, J=1.8 Hz), 7.22 (d,1H, J=8.4 Hz), 7.15-7.14 (m, 2H), 7.09 (d, 1H, J=7.8 Hz), 6.94 (d, 1H,J=8.4 Hz), 6.48 (d, 1H, J=2.4 Hz), 6.42 (d, 1H, J=2.4 Hz), 5.47-5.43 (m,5H), 5.39 (d, 1H, J=5.4 Hz), 5.14 (d, 1H, J=6.0 Hz), 5.10 (d, 1H, J=9.0Hz), 4.59 (br s, 1H), 4.39 (d, 1H, J=7.2 Hz), 4.31-4.26 (m, 2H),3.88-3.82 (m, 2H), 3.77-3.74 (m, 1H), 3.71-3.62 (m, 2H), 3.54 (dd, 1H,J=9.0, 9.0 Hz), 3.13-3.06 (m, 1H), 2.87-2.84 (m, 1H), 2.82 (s, 3H),2.76-2.74 (m, 1H), 2.64-2.61 (m, 1H), 2.33-2.30 (m, 1H), 2.10-2.06 (m,2H), 1.98-1.96 (m, 1H), 1.80 (dd, 1H, J=7.2, 7.2 Hz), 1.64-1.62 (m, 3H),1.56 (s, 3H), 1.31 (s, 1H), 1.22 (d, 3H, J=6.6 Hz), 0.97 (d, 3H, J=6.0Hz), 0.92 (d, 3H, J=6.0 Hz); ESI-TOF HRMS m/z 1434.4581 (M+H⁺,C₆₆H₇₇Cl₂N₉O₂₃ requires 1434.4582.

A solution of Compound 16 (0.68 mg, 0.48 μmol) in anhydrous DMF (60 μL)was treated with 4-(4′-chlorophenyl)benzaldehyde (0.1 M in DMF, 7.2 μL,0.72 μmol) and i-Pr₂NEt (distilled, 0.1 M in DMF, 24.0 μL, 2.4 μmol) at25° C. The reaction mixture was stirred for 2 hours at 50° C. After thereaction was complete, the mixture was treated with NaCNBH₃ (1 M in THF,47.4 μL, 47.4 μmol) and stirred for 5 hours at 70° C.

The reaction mixture was quenched by the addition of 50% CH₃OH in H₂O(0.2 mL) at 25° C. and the residue was purified by semi-preparativereverse-phase HPLC (Zorbax® SB-C18, 5 μm, 9.4×150 mm, 1-40%MeCN/H₂O-0.07% TFA gradient over 40 minutes, 3 mL/minute, t_(R)=34.2minutes) to afford Compound 17 (0.31 mg, 41% yield) as a white amorphoussolid: ¹H NMR (CD₃OD, 600 MHz, 298 K) δ 8.07 (s, 1H), 8.02 (d, 1H, J=7.8Hz), 7.98 (s, 1H), 7.92 (d, 1H, J=7.2 Hz), 7.79 (d, 1H, J=9.0 Hz), 7.76(d, 1H, J=7.2 Hz), 7.70 (d, 2H, J=8.4 Hz), 7.63 (d, 2H, J=8.4 Hz), 7.56(d, 2H, J=8.4 Hz), 7.47 (d, 2H, J=8.4 Hz), 7.43 (s, 1H), 7.33 (s, 1H),7.22 (d, 1H, J=9.0 Hz), 7.12 (br s, 1H), 6.95-6.90 (m, 1H), 6.48 (d, 1H,J=2.4 Hz), 6.40 (d, 1H, J=2.4 Hz), 5.55 (d, 1H, J=4.2 Hz), 5.49-5.46 (m,3H), 5.44-5.34 (m, 2H), 4.37-4.31 (m, 2H), 4.20-4.16 (m, 2H), 4.13-4.07(m, 2H), 3.88-3.82 (m, 2H), 3.74-3.72 (m, 1H), 3.68-3.61 (m, 1H),3.54-3.50 (m, 2H), 3.00 (s, 1H), 2.86 (s, 1H), 2.83 (s, 3H), 2.71-2.64(m, 1H), 2.55-2.50 (m, 1H), 2.23 (dd, 1H, J=7.8, 7.8 Hz), 2.22-2.12 (m,2H), 1.93 (s, 1H), 1.86-1.77 (m, 2H), 1.74 (s, 3H), 1.27 (d, 3H, J=6.6Hz), 1.00 (d, 3H, J=6.0 Hz), 0.95 (d, 3H, J=6.6 Hz); ESI-TOF HRMS m/z817.7525 (M+2H⁺, C₇₉H₈₆Cl₃N₉O₂₃ requires 817.7524).

In a total volume of 10.3 mL, 2.0 mM UDP-glucose (21.7 mg,Sigma-Aldrich, 38.3 μmol) and 0.5 mM Compound 8 [Xie et al., J Am ChemSoc 2012 134:1284-1297 Compound 44] (5.5 mg, 4.8 μmol) were incubatedwith 75 mM Tricine-NaOH (pH 9.0), 2 mM tris(2-carboxyethyl)-phosphine, 1mM MgCl₂, glycerol (25% v/v) and 25 μM GtfE for 38 hours at 37° C. Thereaction mixture was quenched by the addition of MeOH (90 mL) and theresidue was passed through a 0.45 μm polyethersulfone membrane filterand concentrated by evaporation to a final volume of about 2 mL. Afterthe addition of H₂O (1.0 mL), the mixture was purified bysemi-preparative reverse-phase HPLC (Vydac 218TP1022-C18, 10 μm, 22×250mm, 1-40% MeCN/H₂O-0.07% TFA gradient over 40 minutes, 10 mL/minute,t_(R)=22.7 minutes) to afford Compound 20 (2.3 mg, 35%) as a whiteamorphous solid and recovered starting material (2.5 mg, 46%) as a whiteamorphous solid.

¹H NMR (CD₃OD, 600 MHz, 298 K) δ 8.35 (d, 1H, J=6.6 Hz), 7.67-7.66 (m,2H), 7.64-7.60 (m, 2H), 7.35-7.30 (m, 2H), 7.18 (d, 1H, J=2.4 Hz), 6.74(d, 1H, J 9.0 Hz), 6.65 (d, 1H, J=2.4 Hz), 6.44-6.43 (m, 1H), 6.41 (d,1H, J=2.4 Hz), 6.16 (s, 1H), 5.86 (s, 1H), 5.38 (d, 1H, J=7.8 Hz),5.32-5.29 (m, 3H), 5.26 (br s, 1H), 4.40 (d, 1H, J=6.6 Hz), 4.30-4.27(m, 1H), 4.23-4.21 (m, 2H), 4.04-4.01 (m, 2H), 3.97-3.95 (m, 1H),3.88-3.86 (m, 1H), 3.77-3.74 (m, 1H), 3.64-3.62 (m, 1H), 3.50-3.48 (m,2H), 3.42-3.38 (m, 3H), 3.34 (s, 1H), 3.19-3.18 (m, 2H), 3.05-3.00 (m,1H), 2.75 (s, 3H), 2.30-2.22 (m, 1H), 1.90-1.85 (m, 1H), 1.76-1.65 (m,3H), 1.46-1.40 (m, 1H), 1.38-1.36 (m, 1H), 1.00 (d, 3H, J=6.0 Hz), 0.97(d, 3H, J=6.0 Hz); ESI-TOF HRMS m/z 1307.3420 (M+H⁺, C₅₉H₆₅Cl₂N₈O₂₀Srequires 1307.3407).

In a total volume of 1.4 mL, 3.0 mM UDP-vancosamine (2.5 mg, 4.6 μmol)and 0.5 mM Compound 20 (1.1 mg, 0.84 μmol) were incubated with 75 mMTricine-NaOH (pH 9.0), 2 mM tris-(2-carboxyethyl)-phosphine, 0.2 mg/mLbovine serum albumin, 1 mM MgCl₂, glycerol (10% v/v) and 10 μM GtfD for3 hours at 37° C. The reaction mixture was quenched by the addition ofMeOH (10 mL) at 0° C. and was passed through a 0.45 μm polyethersulfonemembrane filter and concentrated by evaporation to a final volume ofabout 2 mL. After the addition of H₂O (2.0 mL), the mixture was purifiedby semi-preparative reverse-phase HPLC (Vydac® 218TP1022-C18, 10 μm,22×250 mm, 1-40% MeCN/H₂O-0.07% TFA gradient over 40 minutes, 10mL/minute, t_(R)=20.7 minutes) to afford Compound 24 (1.0 mg, 84%) as awhite amorphous solid: ¹H NMR (CD₃OD, 600 MHz, 298 K) δ 8.29 (s, 1H),7.73-7.62 (m, 3H), 7.57 (d, 1H, J=9.0 Hz), 7.30 (d, 1H, J=9.0 Hz), 7.26(d, 1H, J=8.4 Hz), 7.18 (s, 1H), 6.96 (d, 1H, J=9.0 Hz), 6.79 (d, 1H,J=8.4 Hz), 6.65 (s, 1H), 6.46-6.42 (m, 3H), 6.07 (s, 1H), 5.77 (s, 1H),5.44 (d, 1H, J=7.8 Hz), 5.40 (d, 1H, J=4.2 Hz), 5.31 (s, 1H), 5.28-5.25(m, 3H), 4.40-4.39 (br m, 1H), 4.31-4.28 (m, 1H), 4.22-4.20 (br m, 2H),4.07-4.01 (m, 2H), 3.97-3.94 (m, 1H), 3.86 (d, 1H, J=12.0 Hz), 3.82-3.79(m, 1H), 3.76-3.73 (m, 1H), 3.68-3.55 (m, 3H), 3.52-3.50 (m, 1H),2.97-2.94 (m, 1H), 2.76 (s, 3H), 2.07-2.04 (m, 1H), 1.92 (d, 1H, J=13.8Hz), 1.88-1.83 (m, 1H), 1.48 (s, 3H), 1.40-1.29 (m, 2H), 1.19 (d, 3H,J=6.6 Hz), 1.02 (d, 3H, J=6.6 Hz), 1.00 (d, 3H, J=6.6 Hz); ESI-TOF HRMSm/z 1450.4375 (M+H⁺, C₆₆H₇₈Cl₂N₉O₂₂S requires 1450.4353).

A mixture of Compound 24 (0.38 mg, 0.26 Mmol) and AgOAc (0.43 mg, 2.6μmol) was treated with anhydrous saturated NH₃—CH₃OH (0.2 mL) at 25° C.The reaction mixture was stirred for 6 hours at 25° C. before thesolvent was removed under a stream of N₂. The residue was dissolved in50% CH₃OH in H₂O (0.2 mL) and purified by semi-preparative reverse-phaseHPLC (Zorbax® SB-C18, 5 μm, 9.4×150 mm, 1-40% MeCN/H₂O-0.07% TFAgradient over 40 minutes, 3 mL/minute, t_(R)=16.4 minutes) to affordCompound 25 (86 μg, 50% yield brsm, unoptimized) as a white film: ¹H NMR(CD₃OD, 600 MHz, 298 K) δ 7.74 (d, J=9.0 Hz, 1H), 7.74 (s, 1H), 6.90 (d,J=8.4 Hz, 1H), 6.68 (s, 1H), 6.44 (s, 1H), 5.52 (d, J=11.2 Hz, 1H),5.45-5.37 (m, 5H), 4.33 (s, 1H), 4.13-4.06 (m, 2H), 3.85 (s, 1H), 3.77(d, J=9.0 Hz, 1H), 3.67-3.52 (m, 2H), 2.88 (s, 3H), 2.45-2.41 (m, 1H),2.07 (d, J=10.8 Hz, 1H), 1.86 (s, 1H), 1.61 (s, 1H), 1.51-1.39 (m, 3H),1.30 (s, 1H), 1.28-1.20 (m, 3H), 0.89 (d, J=6.0 Hz, 3H), 0.85 (d, J=6.0Hz, 3H); ESI-TOF HRMS m/z 1433.4760 (M+H⁺, C₆₆H₇₈Cl₂N₁₀O₂₂ requires1433.4742).

A solution of hydroxymethylvancomycin [Nakayama et al., Org. Lett. 2014,16, 3572] (1.0 mg, 0.65 μmol) in anhydrous DMF (0.1 mL) was treated with4-(4′-chlorophenyl)benzaldehyde (0.1 M in DMF, 9.7 μL, 0.97 μmol) andi-Pr₂NEt (distilled, 0.1 M in DMF, 32.3 μL, 3.23 μmol) at 25° C. Thereaction mixture was stirred for 12 hours at 30° C. After the reactionwas complete, the mixture was treated with NaBH(OAc)₃ (13.7 mg, 64.6μmol) and stirred for 2 hours at 30° C. The reaction mixture wasquenched by the addition of 50% CH₃OH in H₂O (0.2 mL) at 25° C. and theresidue was purified by semi-preparative reverse-phase HPLC (Zorbax®SB-C18, 5 μm, 9.4×150 mm, 1-40% MeCN/H₂O-0.07% TFA gradient over 40minutes, 3 mL/minute, t_(R)=35.2 minutes) to afford Compound 26 (0.73mg, 67% yield) as a white amorphous solid: ¹H NMR (CD₃OD, 600 MHz, 298K)δ 7.71-7.68 (m, 3H), 7.66-7.60 (m, 5H), 7.57-7.54 (m, 2H), 7.47-7.43 (m,3H), 7.32 (d, 1H, J=8.4 Hz), 7.29 (d, 1H, J=7.8 Hz), 7.11 (d, 1H, J=8.4Hz), 6.94 (d, 1H, J=8.4 Hz), 6.45-6.43 (m, 1H), 6.41-6.37 (m, 1H), 6.35(d, 1H, J=2.4 Hz), 6.33 (d, 1H, J=2.4 Hz), 6.41-6.37 (m, 1H), 6.34-6.33(m, 1H), 5.74 (d, 1H, J=11.2 Hz), 5.60 (d, 1H, J=18.0 Hz), 5.56 (d, 1H,J=7.8 Hz), 5.53 (d, 1H, J=12.0 Hz), 5.49 (d, 1H, J=4.2 Hz), 5.44-5.42(m, 1H), 4.55-4.53 (m, 1H), 4.22-4.15 (m, 1H), 4.13-4.05 (m, 1H),3.95-3.93 (m, 1H), 3.86-3.84 (m, 2H), 3.75-3.72 (m, 1H), 3.65 (br s,1H), 3.62 (d, 1H, J=9.6 Hz), 3.53-3.50 (m, 1H), 2.78 (s, 3H), 2.52-2.48(m, 1H), 2.21-2.16 (m, 1H), 2.04 (d, 1H, J=13.2 Hz), 1.67-1.64 (br m,5H), 1.32 (d, 3H, J=6.6 Hz), 1.03-1.00 (m, 3H), 0.98-0.95 (m, 3H);ESI-TOF HRMS m/z 1634.5057 (M+H⁺, C₇₉H₈₇Cl₃N₉O₂₃ requires 1634.4975).

This reaction was run on scales of 0.2-1.1 mg (67-74%) as part of theoptimization of conditions for use with Compound 27 on the amountsavailable.

A solution of Compound 24 (0.62 mg, 0.42 μmol) in anhydrous DMF (30 μL)was treated with 4-(4′-chlorophenyl)benzaldehyde (0.1 mM in DMF, 5.5 μL,0.546 μmol) and i-Pr₂NEt (distilled, 0.1 mM in DMF, 21 μL, 2.1 μmol) at25° C. The reaction mixture was stirred for 9 hours at 30° C. After thereaction was complete, the mixture was treated with NaBH(OAc)₃ (11.2 mg,42.0 μmol) and stirred for 2 hours at 30° C.

The reaction mixture was quenched with the addition of 50% CH₃OH in H₂O(0.2 mL) and the residue was purified by semi-preparative reverse-phaseHPLC (1-40% MeCN/H₂O-0.07% TFA isocratic gradient over 40 minutes) toafford Compound 27 (0.52 mg, 74%) as a white amorphous solid: ¹H NMR(CD₃OD, 600 MHz) δ 8.30 (d, 1H, J=6.6 Hz), 7.72-7.67 (m, 5H), 7.63 (d,2H, J=8.4 Hz), 7.60 (d, 1H, J=6.0 Hz), 7.56 (d, 2H, J=7.8 Hz), 7.47 (d,2H, J=8.4 Hz), 7.33 (d, 1H, J=8.4 Hz), 7.29 (d, 1H, J=8.4 Hz), 7.19 (d,1H, J=2.4 Hz), 6.99-6.97 (m, 1H), 6.80 (d, 1H, J=8.4 Hz), 6.56 (d, 1H,J=2.4 Hz), 6.42 (d, 1H, J=1.8 Hz), 6.13-6.08 (br m, 1H), 5.80 (s, 1H),5.51 (d, 1H, J 7.2 Hz), 5.46 (d, 1H, J=4.8 Hz), 5.33 (d, 1H, J=3.0 Hz),5.31-5.29 (br m, 2H), 5.27 (s, 1H), 4.40-4.39 (m, 1H), 4.33-4.30 (m,1H), 4.24 (s, 1H), 4.16 (d, 1H, J=12.0 Hz), 4.08-4.02 (m, 3H), 3.98-3.95(m, 1H), 3.90-3.82 (m, 2H), 3.77-3.75 (m, 1H), 3.64-3.60 (m, 2H),3.54-3.50 (m, 2H), 3.44-3.42 (m, 1H), 3.20-3.19 (m, 1H), 2.97 (d, 1H,J=13.8 Hz), 2.77 (s, 3H), 2.36-2.32 (m, 1H), 2.20-2.16 (m, 1H), 2.02 (d,1H, J=13.8 Hz), 1.89-1.84 (m, 1H), 1.81-1.76 (m, 1H), 1.71-1.68 (m, 1H),1.66 (s, 3H), 1.27 (d, 3H, J=6.6 Hz), 1.03 (d, 3H, J=6.0 Hz), 1.00 (d,3H, J=6.6 Hz); ESI-TOF HRMS m/z 825.7447 (M+2H⁺, C₇₉H₈₆Cl₃N₉O₂₃ requires825.7410).

A mixture of Compound 27 (0.35 mg, 0.20 μmol) and AgOAc (0.33 mg, 2.0μmol) was treated with anhydrous saturated NH₃—CH₃OH (0.2 mL) at 25° C.The reaction mixture was stirred for 6 hours at 25° C. before thesolvent was removed under a stream of N₂. The residue was dissolved in50% CH₃OH in H₂O (0.2 mL) and purified by semi-preparative reverse-phaseHPLC (Zorbax® SB-C18, 5 μm, 9.4×150 mm, 1-40% MeCN/H₂O-0.07% TFAgradient over 40 minutes, 3 mL/minute, t_(R)=332 minutes) to affordCompound 28 (86 μg, 48% yield brsm, unoptimized) as a white film: ¹H NMR(CD₃OD, 600 MHz, 298 K) δ 7.79-7.68 (m, 3H), 7.61 (d, J=8.4 Hz, 2H),7.54 (d, J=7.8 Hz, 2H), 7.46-7.44 (m, 4H), 7.08-7.02 (br m, 2H), 6.88(d, J=9.0 Hz, 1H), 6.66 (s, 1H), 6.43 (d, J=2.4 Hz, 1H), 5.58-5.48 (m,2H), 5.44-5.40 (m, 3H), 4.37-4.28 (m, 1H), 4.18-4.15 (m, 2H), 4.11 (d,J=4.2 Hz, 1H), 4.09-4.02 (m, 4H), 3.88-3.75 (m, 2H), 3.67-3.54 (m, 4H),2.87 (s, 3H), 2.74 (br s, 1H), 2.41 (dd, J=14.4, 4.8 Hz, 1H), 2.19-2.16(m, 1H), 2.06 (s, 1H), 2.03 (s, 1H), 1.85 (br s, 1H), 1.65-1.55 (m, 4H),1.30-1.29 (m, 4H), 1.22-1.19 (m, 1H), 0.88 (d, J=6.0 Hz, 3H), 0.84 (d,J=6.6 Hz, 3H); ESI-TOF HRMS m/z 817.2614 (M+2H⁺, C₇₉H₈₈Cl₃N₁₀O₂₂requires 817.2604).

This compound was described in Nakayama et al., Org. Lett. 2014,16:3572.

This compound was described in Okano et al., J. Am. Chem. Soc. 2014,136, 13522.

Antimicrobial Assays

Assays were carried out following the methods of Clinical and LaboratoryStandards Institute; Methods for Dilution Antimicrobial SusceptibilityTests for Bacteria That Grow Aerobically; Approved Standard, 7th ed.;CLSI document M07-A8; Clinical and Laboratory Standards Institute:Wayne, Pa., 2009.

One day before studies were carried out, fresh cultures ofvancomycin-sensitive Staphylococcus aureus (VSSA strain ATCC 25923),methicillin and oxacillin-resistant Staphylococcus aureus subsp. aureus(MRSA strain ATCC 43300), vancomycin-resistant Enterococcus faecalis(VanA VRE, BM4166), Enterococcus faecium (VanA VRE, ATCC BAA-2317) andvancomycin-resistant Enterococcus faecalis (VanB VRE, strain ATCC51299), were inoculated and grown in an orbital shaker at 37° C. in 100%Mueller-Hinton broth (VSSA, MRSA and VanB VRE) or 100% Brain-HeartInfusion broth (VanA VRE). After 24 hours, the bacterial stock solutionswere serial diluted with the culture medium (10% Mueller-Hinton brothfor VSSA, MRSA and VanB VRE or 10% Brain-Heart Infusion broth for VanAVRE) to achieve the turbidity equivalent of 1:100 dilution of 0.5 MMacfarland solution. This diluted bacterial stock solution was theninoculated into a well of a V-shaped 96-well glass coated microtiterplate, supplemented with serial diluted aliquots of the antibioticsolution DMSO (4 μL), to achieve a total assay volume of 0.1 mL. Theplate was then incubated at 37° C. for 18 hours, after which minimalinhibitory concentrations (MICs) were determined by monitoring the cellgrowth (observed as a pellet) in the wells. The lowest concentration ofantibiotic (in μg/mL) capable of eliminating the cell growth in thewells is reported as the MIC. The reported MIC values for the newantibiotics were determined against vancomycin as a standard in thefirst well, which have well-established MIC values.

Each of the patents, patent applications and articles cited herein isincorporated by reference.

The foregoing description and the examples are intended as illustrativeand are not to be taken as limiting. Still other variations within thespirit and scope of this invention are possible and will readily presentthemselves to those skilled in the art.

What is claimed:
 1. A compound that corresponds in structure to that shown in Formula I or its pharmaceutically acceptable salt,

wherein X=S or NH; and R is selected from the group consisting of (C₁-C₁₆)hydrocarbyl, aryl(C₁-C₆)-hydrocarbyldiyl, heteroaryl-(C₁-C₆)hydrocarbyldiyl, (C₁-C₆)hydrocarbyldiylheteroaryl, halo(C₁-C₁₂)-hydrocarbyldiyl, and (C₁-C₁₆)amido substituents, wherein an aryl or heteroaryl group is itself optionally substituted with up to three substituents independently selected from the group consisting of: (i) hydroxy, (ii) halo, (iii) nitro, (iv) (C₁-C₆)hydrocarbyl, (v) halo(C₁-C₁₆)hydrocarbyl, (vi) (C₁-C₆)hydrocarbyloxy, (vii) halo(C₁-C₆)hydrocarbyloxy, (viii) aryl, and (ix) aryloxy, wherein an aryl or aryloxy substituent can itself be substituted with up to three substituents independently selected from the group consisting of: (i) hydroxy, (ii) halo, (iii) nitro, (iv) (C₁-C₆)hydrocarbyl, (v) halo(C₁-C₁₆)hydrocarbyl, (vi) (C₁-C₆)hydrocarbyloxy, and (vii) halo(C₁-C₆)hydrocarbyloxy; and R¹ is CH₂OH, CH₂OR², where R² is (C₁-C₇)hydrocarboyl, C(O)OH [carboxyl], C(O)R³, where R³ is (C₁-C₆)hydrocarbyloxy, or NR⁴R⁵ where R⁴ and R⁵ are independently the same or different and are H, (C₁-C₆)hydrocarbyl or R⁴ and R⁵ together with the depicted nitrogen atom form a 5-7 membered ring that can contain one ring oxygen atom.
 2. The compound or its pharmaceutically acceptable salt according to claim 1, wherein R is aryl(C₁-C₆)-hydrocarbyldiyl.
 3. The compound or its pharmaceutically acceptable salt according to claim 2 that corresponds in structure to Formula II,


4. The compound or its pharmaceutically acceptable salt according to claim 2 that corresponds in structure to Formula III,


5. The compound or its pharmaceutically acceptable salt according to claim 2, wherein said aryl(C₁-C₆)-hydrocarbyldiyl R group is a 4-(4′-chlorophenyl)phenylmethyldiyl group.
 6. The compound or its pharmaceutically acceptable salt according to claim 2 that corresponds in structure to one or more of the formulas below


7. The compound or its pharmaceutically acceptable salt according to claim 2 that corresponds in structure to one or more of the formulas below


8. A pharmaceutical composition that comprises an antimicrobial amount of a compound of Formula I or a pharmaceutically acceptable salt thereof dissolved or dispersed in a physiologically acceptable diluent

wherein X=NH; and R is selected from the group consisting of (C₁-C₁₆)hydrocarbyl, aryl(C₁-C₆)hydrocarbyldiyl, heteroaryl(C₁-C₆)hydrocarbyldiyl, (C₁-C₆)hydrocarbyldiylheteroaryl, halo(C₁-C₁₂)-hydrocarbyldiyl, and (C₁-C₁₆)amido substituents, wherein an aryl or heteroaryl group is itself optionally substituted with up to three substituents independently selected from the group consisting of: (i) hydroxy, (ii) halo, (iii) nitro, (iv) (C₁-C₆)hydrocarbyl, (v) halo(C₁-C₁₆)hydrocarbyl, (vi) (C₁-C₆)hydrocarbyloxy, (vii) halo(C₁-C₆)hydrocarbyloxy, (viii) aryl, and (ix) aryloxy, wherein an aryl or aryloxy substituent can itself be substituted with up to three substituents independently selected from the group consisting of: (i) hydroxy, (ii) halo, (iii) nitro, (iv) (C₁-C₆)hydrocarbyl, (v) halo(C₁-C₁₆)hydrocarbyl, (vi) (C₁-C₆)hydrocarbyloxy, and (vii) halo(C₁-C₆)hydrocarbyloxy; and R¹ is CH₂OH, CH₂OR², where R² is (C₁-C₇)hydrocarboyl, C(O)OH [carboxyl], C(O)R³, where R³ is (C₁-C₆)hydrocarbyloxy, or NR⁴R⁵ where R⁴ and R⁵ are independently the same or different and are H, (C₁-C₆)hydrocarbyl or R⁴ and R⁵ together with the depicted nitrogen atom form a 5-7 membered ring that can contain one ring oxygen atom.
 9. The pharmaceutical composition according to claim 8, wherein R is aryl(C₁-C₆)-hydrocarbyldiyl.
 10. The pharmaceutical composition according to claim 9, wherein said aryl(C₁-C₆)-hydrocarbyldiyl R group is a 4-(4′-chlorophenyl)phenylmethyldiyl group.
 11. The pharmaceutical composition according to claim 10, wherein the dissolved or dispersed compound or a pharmaceutically acceptable salt thereof is one or more of


12. A compound or its pharmaceutically acceptable salt that corresponds in structure to one or both of the formulas below


13. A pharmaceutical composition that comprises an antimicrobial amount of a compound or a pharmaceutically acceptable salt thereof according to claim 12 dissolved or dispersed in a physiologically acceptable diluent. 